High efficient production of Pr55gag virus-like particles expressing multiple HIV-1 epitopes, including a gp120 protein derived from an Ugandan HIV-1 isolate of subtype A
Introduction
The development of an effective, safe and affordable vaccine strategy represents a crucial goal for both industrialized and developing countries. Different candidate HIV-1 vaccine strategies have been developed since early 90s, including whole inactivated virus (Daniel et al., 1992, Almond et al., 1995, Cranage et al., 1997), recombinant HIV-1 envelope subunits (Berman et al., 1990, Girard et al., 1991, Fultz et al., 1992, el-Amad et al., 1995), live vector-based (Berglund et al., 1997, Girard et al., 1997) and DNA-based vaccines (Boyer et al., 1997, Letvin et al., 1997).
In order to develop a vaccine model characterized by the capacity of inducing a strong humoral as well as cellular immune response, without risks of pathogenicity, we have chosen an approach based on virus-like particles (VLPs). The HIV-1 Pr55gag precursor protein, when expressed in recombinant baculovirus-infected cells, assembles as immature, non-replicating and non-infectious HIV–VLPs (Gheysen et al., 1989, Wagner et al., 1994). This can be used as particulate antigen delivery system, able to induce a significant immune response against epitopes packaged in the resulting particles. In particular, VLPs have been employed to deliver linear epitopes or complete conformational proteins, with an effective induction of both arms of the immune response (Haffar et al., 1991, Rovinski et al., 1992, Griffiths et al., 1993, Wagner et al., 1994, Osterrieder et al., 1995, Deml et al., 1997a, Tobin et al., 1997).
In this perspective, different approaches have been employed to package HIV-1 envelope epitopes into VLPs in order to induce neutralizing antibodies. In particular, immunologically relevant sequences have been introduced into the Pr55gag precursor (Haffar et al., 1990, Wagner et al., 1994) or fused to the 3′ end of the gag ORF, taking advantage of the ribosomal frameshifting signals (Tobin et al., 1997). Moreover, the complete gp120 envelope protein has been packaged into the VLP in order to express conformational epitopes. In particular, the gp120 molecule has been anchored on the Pr55gag-VLPs through the trans-membrane (TM) portion of the Epstein–Barr virus (EBV) gp220/350, to increase the expression and stability of the chimeric gp120-TM on the VLPs surface (Deml et al., 1997a). Chemical cross-linking analyses suggest that these gp120-TM molecules retain the ability to form oligomeric structures (Deml et al., 1997a), which seem to be a conformational requirement for the induction of cross-neutralizing antibodies (Sattentau and Moore, 1995, Fouts et al., 1997). Such envelope-presenting HIV–VLPs have shown a significant immunogenicity in Balb/c mouse and Rhesus Macaque models, with the induction of both neutralizing antibodies and CTLs in absence of adjuvants (Deml et al., 1997b, Wagner et al., 1998). Moreover, the VLP model is currently under investigation as potential vaccine for other human and non-human viruses, such as hepatitis, papilloma, pseudorabies and equine herpes viruses (Kirnbauer et al., 1992, Garnier et al., 1995, Osterrieder et al., 1995, Li et al., 1997).
Based on these extensive observations on the feasibility and efficacy of the VLP model as a potential preventive vaccine, we investigated a novel approach to obtain a more efficient packaging of multiple epitopes/proteins in the VLP structures. Our intent was to increase the transposition frequency of multiple sequences on the same baculovirus genomic DNA, in a prokaryotic system, and to find a more reliable strategy of screening the recombinants, in order to avoid the homologous recombination step in insect cells with multiple rounds of plaque purification. The approach described in this study is based on a commercially available baculovirus expression system (Gibco-BRL), which allows the independent expression of multiple genes from the same transfer vector. A site-specific transposition of viral genes into the baculovirus DNA is then obtained within a modified bacterial strain and the generation of recombinant baculovirus DNA is coupled to the identification of colorless bacterial colonies.
The latest data produced by the ‘UNAIDS HIV Network’ indicate that the A- and C-clades account for 73% (25 and 48%, respectively) of all HIV-1 isolates identified worldwide, implying that, in order to have better chances of efficacy, future vaccine strategies should be based on a cocktail of antigens derived from such clades. This prompted us to package into the VLP a gp120UG derived from an Ugandan HIV-1 isolate of the A-clade (UG5.94UG018), previously characterized in our Laboratory (Buonaguro et al., 1995, Buonaguro et al., 1998a, Buonaguro et al., 1998b).
Furthermore, the possibility has been verified of packaging into the VLPs the chimeric gp120UG-TM molecule as well as the Nef and Pol proteins which show a much lower divergence among HIV-1 strains and could induce cross-clade immunity.
Our results clearly indicate that multiple epitope/proteins can be included into the VLP structure without affecting the Pr55gag auto-assembling properties and, therefore, that Pr55gag-based VLPs can provide a ductile antigenic presentation model for developing candidate anti-HIV-1 vaccines.
Section snippets
Cloning of virus genes in the transfer vector
The trans-membrane (TM) fragment of the EBV glycoprotein gp220/350, consisting of 22 aa of the trans-membrane domain and 27 aa of the cytoplasmic region, was linked to the C-terminus of the gp120 coding region via a Ser-Gly-Ser-Gly-Ala-Gly flexible hinge, as previously described (Deml et al., 1997a). In particular, the gp120 reading frame from the 94UG018 Ugandan HIV-1 A-clade isolate (Buonaguro et al., 1998b) was PCR amplified by the primer set A (5′-CGTCGCAGAAGACAGTGGCAATGA-3′) and B
V3 amino acid sequence of the 94UG018 isolate
The 94UG018 strain has been chosen among 10 HIV-1 Ugandan isolates, previously identified by us in a rural area of Northern Uganda within a cohort of pregnant women (Buonaguro et al., 1995).
The 94UG018 shows a 12.6% average nucleotide divergence with gp120s of the A-clade and clusters with Ugandan/Rwandan samples in a major subgroup with a very strong phylogenetic relationship (97% of bootstrap value) (Buonaguro et al., 1998b).
Furthermore, the V3 loop amino acid sequence shows an 82.9–100%
Discussion
The main objective of this study was to engineer a Virus-like Particle model, based on the HIV-1 Pr55gag and produced in a Baculovirus expression system, presenting a broad spectrum of HIV-1 epitopes/proteins. The nucleotide sequence of an entire gp120, derived from an Ugandan HIV-1 isolate of the A-clade (94UG018), has been analyzed in our laboratory and the derived amino acid sequence of the V3 region shows an 88.5% average homology with isolates of clade-A and a 94.3% homology (33 out of 35
Acknowledgements
This study was supported by grants from the Ministero Italiano della Sanità (Ricerca Corrente and Progetto Finalizzato AIDS 1999 Contract No. 40B.46) and the ICSC-World Lab, Lausanne, Switzerland (Project MCD-2/7). The GenBank accession number for the gp120 nucleotide sequence reported in this paper is AF062521. We thank John McKnight for revising the English style of the manuscript.
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