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. 2004 May;78(9):4710-9.
doi: 10.1128/jvi.78.9.4710-4719.2004.

Elicitation of neutralizing antibodies with DNA vaccines expressing soluble stabilized human immunodeficiency virus type 1 envelope glycoprotein trimers conjugated to C3d

Joseph F Bower  1 , Xinzhen Yang, Joseph Sodroski, Ted M Ross
Affiliations

Elicitation of neutralizing antibodies with DNA vaccines expressing soluble stabilized human immunodeficiency virus type 1 envelope glycoprotein trimers conjugated to C3d

Joseph F Bower et al. J Virol. 2004 May.

Abstract

DNA vaccines expressing the envelope (Env) of human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting immune responses. Oligomeric or trimeric (gp140) forms of Env that more closely mimic the native proteins on the virion are often more effective immunogens than monomeric (gp120) envelopes. In this study, several forms of Env constructed from the HIV-1 isolate YU-2 (HIV-1(YU-2)) were tested for their immunogenic potential: a trimeric form of uncleaved (-) Env stabilized with a synthetic trimer motif isolated from the fibritin (FT) protein of the T4 bacteriophage, sgp140(YU-2)(-/FT), was compared to sgp140(YU-2)(-) without a synthetic trimerization domain, as well as to monomeric gp120(YU-2). DNA plasmids were constructed to express Env alone or fused to various copies of murine C3d (mC3d). BALB/c mice were vaccinated (day 1 and week 4) with DNA expressing a codon-optimized envelope gene insert, alone or fused to mC3d. Mice were subsequently boosted (week 8) with the DNA or recombinant Env protein. All mice had high anti-Env antibody titers regardless of the use of mC3d. Sera from mice vaccinated with DNA expressing non-C3d-fused trimers elicited neutralizing antibodies against homologous HIV-1(YU-2) virus infection in vitro. In contrast, sera from mice inoculated with DNA expressing Env-C3d protein trimers elicited antibody that neutralized both homologous HIV-1(YU-2) and heterologous HIV-1(ADA), albeit at low titers. Therefore, DNA vaccines expressing trimeric envelopes coupled to mC3d, expressed in vivo from codon-optimized sequences, elicit low titers of neutralizing antibodies against primary isolates of HIV-1.

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Figures

FIG. 1.
FIG. 1.
Schematic representation of the gene inserts used to construct each of the vaccine plasmids. Plasmids expressing HIV-1YU-2 envelope gene inserts sgp120YU-2 (A), sgp140YU-2 (B), and sgp140YU-2(−/FT) (C), were constructed, and a representative of how the individual Envs assemble as either monomers or trimers is shown. A schematic representation of the addition of one, two, and three copies of mC3d fused to the 3′ end of each HIV-1YU-2 envelope gene insert, sgp120YU-2 (A), sgp140YU-2 (B), or sgp140YU-2(−/FT) (C), is shown. Each of the gene inserts was cloned into the previously described eukaryotic vaccine vector pTR600 (30, 50, 51). The vector contains the CMV immediate-early promoter (CMV promoter) plus intron A for efficiently initiating transcription of eukaryotic inserts and the bovine growth hormone polyadenylation terminator for efficient termination of transcription. This vector also contains the Col E1 origin of replication for prokaryotic replication as well as the kanamycin resistance (Kanr) gene for selection in antibiotic media.
FIG. 2.
FIG. 2.
Expression of vaccine plasmids was visualized by Western blot analysis. Nonreducing (A) and reducing (B) SDS-PAGE analysis demonstrated that all constructs are efficiently expressed in vitro and FT-stabilized versions of sgp140YU-2(−) form trimers even after boiling and under denaturing conditions with and without the addition of one, two, or three copies of mC3d. Human embryonic kidney cells were transiently transfected with 2 μg of plasmid DNA from each vaccine plasmid. Supernatants were collected, and 1.5% of the total volume was subjected to electrophoresis. Each blot was probed with HIV-Ig followed by anti-human IgG conjugated to horseradish peroxidase and detected with enhanced chemiluminescence. (C) Expression ELISA data to determine the relative level of expression of codon-optimized DNA vaccine plasmids were quantified using 1.5% of the total supernatant from transiently transfected 293T cells and comparing relative amounts to recombinant gp120YU-2 (ImmunoDiagnostics) as a standard and are expressed as micrograms per milliliter of Env secreted into the supernatant. Lane 1, molecular mass marker; lane 2, sgp120YU-2; lane 3, sgp120YU-2 mC3d1; lane 4, sgp120YU-2-mC3d2; lane 5, sgp120YU-2-mC3d3; lane 6, sgp140YU-2(−); lane 7, sgp140YU-2(−)-mC3d1; lane 8, sgp140YU-2(−)-mC3d2; lane 9, sgp140YU-2(−)-mC3d3; lane 10, sgp140YU-2(−/FT); lane 11, sgp140YU-2(−/FT)-mC3d1; lane 12, sgp140YU-2(−/FT)-mC3d2; lane 13, sgp140YU-2(−/FT)-mC3d3.
FIG. 3.
FIG. 3.
Anti-Env IgG raised by sgp120YU-2, sgp140YU-2(−), and sgp140YU-2(-/FT) inoculations was determined by an endpoint dilution titer. BALB/c mice (n = 10 per group) were primed at day 0 with each vaccine construct indicated, sgp120YU-2, sgp140YU-2(−), or sgp140YU-2(−/FT) with or without the addition of one, two, or three copies of mC3d, using 2 μg of DNA inoculated via gene gun and boosting at week 4 with an additional 2 μg of DNA via gene gun. At week 8, each group was split into two and either received a third inoculation of 2 μg of DNA or was boosted with 10 μg of recombinant protein via intraperitoneal injection at two sites of 100 μl with RIBI (Sigma) as adjuvant. Solid symbols represent mice receiving DNA as a third inoculation (A, C, and E); open symbols represent mice receiving recombinant protein as a third inoculation (B, D, and F). Sera were collected prior to and 2 weeks after each inoculation as indicated, and each mouse was assayed for specific IgG levels by ELISA. The 96-well plates were coated with 25 ng of gp120YU-2 recombinant protein. Data are represented as the average of five mice. Preimmune-phase sera from mice had no detectable specific IgG. Endpoint dilution titers were determined by diluting the sera until OD values reached background levels. As shown, all of the constructs elicited similar high-titer Env-specific antibodies regardless of the vaccination regimen used.
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