Modifications of the Human Immunodeficiency Virus Envelope Glycoprotein Enhance Immunogenicity for Genetic Immunization

In this study, we have investigated the effect of specific mutationsin human immunodeficiency virus type 1 (HIV-1) envelope (Env)on antibody production in an effort to improve humoral immuneresponses to this glycoprotein by DNA vaccination. Mice wereinjected with plasmid expression vectors encoding HIV Env withmodifications in regions that might affect this response. Eliminationof conserved glycosylation sites did not substantially enhancehumoral or cytotoxic-T-lymphocyte (CTL) immunity. In contrast,a modified gp140 with different COOH-terminal mutations intendedto mimic a fusion intermediate and stabilize trimer formationenhanced humoral immunity without reducing the efficacy of theCTL response. This mutant, with deletions in the cleavage site,fusogenic domain, and spacing of heptad repeats 1 and 2, retainednative antigenic conformational determinants as defined by bindingto known monoclonal antibodies or CD4, oligomer formation, andvirus neutralization in vitro. Importantly, this modified Env,gp140 ${Delta}$ CFI, stimulated the antibody response to native gp160 whileit retained its ability to induce a CTL response, a desirablefeature for an AIDS vaccine.

INTRODUCTION

Top

Introduction

Plasmid DNA vaccination has been a useful technology for thedevelopment and analysis of immunogens. This method of vaccinationallows relevant posttranslational modifications, appropriateintracellular trafficking, and antigen presentation. Directinjection of naked DNA either intramuscularly or intradermallyreadily induces protective immune responses in animal models.Though DNA vaccines readily elicit cell-mediated immune responses,their ability to induce high-titer antibody responses has beenlimited, particularly to human immunodeficiency virus type 1(HIV-1) envelope (Env). However, plasmid expression vectorscan be readily modified to express different forms of HIV envelopeproteins, enabling rapid and systematic testing of alternativevaccine immunogens.

To improve the immune response to native gp160 and to exposethe core protein for optimal antigen presentation and recognition,we have analyzed the immune response to modified forms of theprotein. The conserved N-linked glycosylation sites previouslysuggested to limit the antibody response (39) were comprehensivelyanalyzed. In addition, the important coiled-coil hairpin regioninvolved with formation of fusion intermediates has been studied.Expression vectors with deletions in the cleavage site (C),the fusion peptide (F), and the interspace (I) between the twoheptad repeats, termed ${Delta}$ CFI deletions, were prepared. In thisreport, the immune response to Env candidates expressed in plasmidswith codons modified to improve gene expression has been analyzed.Both antibody and cytotoxic-T-lymphocyte (CTL) responses wereevaluated after injection of plasmid DNA into muscle. A modifiedgp140 DNA has been identified that better elicits antibody responsesat the same time that it retains its capacity to induce CTLresponses to HIV Env. This prototype may facilitate the identificationof immunogens which can elicit broadly neutralizing antibodyresponses to HIV by gene-based vaccination.

MATERIALS AND METHODS

Top

Materials and Methods

Immunogens. Plasmids expressing the CXCR4-tropic HIV-1 HXB2 Env were madesynthetically with sequences designed to disrupt viral RNA structuresthat limit protein expression by using codons typically foundin human cells (1, 37, 38, 41, 42, 47). Briefly, the syntheticenv gene of HXB2 (GenBank accession number K03455) was generatedin three fragments by assembling the overlapping synthetic oligonucleotidesusing PCR amplification. Glycosylation mutants were generatedby site-directed mutagenesis to replace asparagine with glutamicacid residues in a block of either 11 or 17 conserved glycosylationsites between amino acids 88 and 448 (Fig. 1). To produce aCCR5-tropic version of the HIV-1 envelope, the most divergentregion encoding amino acids 275 to 361 of HXB2 (CXCR4-tropic)gp160 from Bal was replaced with CCR5-tropic HIV-1 BaL sequence(GenBank accession number M68893), which includes the V3 loop.To express truncated mutant Env proteins, stop codons were introducedafter positions 752, 704, 680, or 592 to produce gp150, gp145,gp140, or gp128, respectively. The Env protein was further changedby deleting amino acids 503 to 537 and amino acids 593 to 619,which removes the cleavage site sequence, the fusion domain,and a part of the spacer between the two heptad repeats. Allof these mutations were confirmed by sequencing of both strandsof the cDNAs. The structures of the synthetic HIV envelope genesare shown (Fig. 1). The cDNAs were cloned in the expressionvector pVR1012 (56) under the control of the cytomegalovirusimmediate-early enhancer, promoter, and first intron. Sequenceanalysis indicated that the codon-optimized envelope containedthe following minor point substitutions: F53L, N94D, K192S,I215N, A224T, A346D, P470L, T723I, and S745T.

View larger version (26K):
[in this window]
[in a new window]
FIG. 1. Schematic representation of functional domains and mutations in HIV-1 Env glycoproteins. Full-length envelope polyprotein, gp160, with the indicated features based on the amino acid residues of HXB2 is shown (top). Functional domains include the gp120/gp41 cleavage site (residues 510 and 511), the fusion domain (residues 512 to 527), the two heptad repeats (residues 546 to 579 and residues 628 to 655), the transmembrane domain (residues 684 to 705), and the cytoplasmic domain (residues 706 to 856). The mutant forms of the envelope proteins are shown below the structure of gp160. COOH deletions were introduced that terminate the envelope protein at positions 752, 704, or 680 to produce gp150, gp145, or gp140, respectively. Two internal deletions that removed the cleavage site, the fusion domain, and the region between the two heptad repeats were introduced into gp160, gp150, gp145, and gp140. A further deletion in the COOH-terminal region at position 592 removed the second heptad repeat and the transmembrane domain to produce gp128 ${Delta}$ CFI. To disrupt potential glycosylation sites, asparagine (N) residues at 11 positions (88, 156, 160, 197, 230, 234, 241, 262, 276, 289, and 295) were replaced with aspartic acid (D) residues in both gp160 and gp150. Versions of both gp160 and gp150 were created with a total of 17 mutated glycosylation sites by including six additional N-to-D substitutions at positions 332, 339, 356, 386, 392, and 448.

Expression of envelope proteins in transfected cells. 293 cells (10⁶) were plated in 60-mm-diameter dishes. Cellswere transfected on the following day with 2 µg of plasmidusing calcium phosphate (16, 56). Cells were harvested 48 hafter transfection using phosphate-buffered saline (PBS) containing2 mM EDTA (PBS-EDTA). Cells were lysed in buffer containing50 mM HEPES (pH 7.0), 250 mM NaCl, and 0.5% NP-40. The proteinconcentration in the lysates was determined using the Bradfordassay (Bio-Rad, Hercules, Calif.), and the protein (25 µg)was analyzed by separation on 7.5% sodium dodecyl sulfate (SDS)-7.5%polyacrylamide gel and transfer to Immobilon-P membrane (Millipore,Bedford, Mass.). The antibody response to mutant Env was detectedby immunoprecipitation with sera from mice followed by Westernblotting (Fig. 2A, C, D, and E) with polyclonal antibody againstgp160 (Intracel, Rockville, Md.). ß-Actin was usedas an internal control with an antibody to this gene product(Sigma, St. Louis, Mo.). As shown in Fig. 2B, 293 cells weretransfected either with 2 µg of control vector or variousamounts (2.0, 0.5, and 0.05 µg) of vector expressing eitherviral gp160 or codon-altered gp160 using the protocol above.Cells were harvested 48 h after transfection and lysed in buffercontaining 50 mM HEPES (pH 7.0), 250 mM NaCl, and 0.5% NP-40.Processing of Env was detected as described above by Westernblotting using supernatant from a hybridoma producing monoclonalantibody against gp41 (HIV-1 IIIB gp41 hybridoma [chessie 8];NIH AIDS Research and Reference Reagent Program [kindly providedby George Lewis]).

View larger version (37K):
[in this window]
[in a new window]
FIG. 2. Comparison of the expression of the HIV-1 gp160 with codon-optimized gp160. (A and B) Expression of plasmids encoding Rev-dependent and Rev-independent codon-modified gp160 (lanes 1 and 2). (A) The upper panel shows expression of Rev-dependent viral gp160 (left) and codon-modified gp160 (right) in transfected 293 cells. The lower panel shows comparable expression of ß-actin in these transfected cells. (B) Processing of gp160 was detected with a monoclonal antibody to gp41with viral or codon-altered gp160, as indicated (lanes 3 to 9). (C) Expression of mutant CXCR4-tropic HIV Env glycoproteins with COOH-terminal truncations is shown. (D and E) CXCR4-tropic envelope proteins containing mutant glycosylation sites and mutant functional domains are shown. The indicated proteins were detected by immunoblotting as described above. Cell lysates produced by transfection with vector containing no insert were used as controls (vector, first lane in each panel).

Expression of soluble ${Delta}$ CFI HIV-1 envelope variants. 293 cells were transfected either with a control vector withno insert or the same vector expressing gp160, gp145 ${Delta}$ CFI, orgp140 ${Delta}$ CFI as described above. Cell-free media were collected48 h after transfection and clarified by centrifugation at 16,000x g at 4°C. Equal volumes of cell-free media were used forimmunoprecipitation with 1 µg of HIV-1 immunoglobulinG (IgG). The precipitated proteins were separated by SDS-7.5%polyacrylamide gel electrophoresis (PAGE), and Env was detectedby Western blotting using a polyclonal antiserum against gp160(Intracel).

Cell surface expression of envelope by fluorescence-activated cell sorting (FACS) analysis. 293 cells were harvested 48 h after transfection using calciumphosphate ( $~$ 90% transfection efficiency), as in the previoussection, and washed twice with PBS containing 1% bovine serumalbumin and incubated for 30 min on ice with either controlmouse or human IgG or monoclonal 2F5 or 2G12 or polyclonal Igfrom an HIV-1-infected patient. The secondary antibody againstthe relevant human or mouse IgG conjugated with fluoresceinisothiocyanate (Jackson Immuno Research) was added, incubatedfor 30 min on ice, washed three times with PBS, and analyzedby flow cytometry (FACScan). The median fluorescent intensityvalues were derived using Cell Quant software.

CD4 binding assay. The binding assay with soluble CD4 (sCD4) was performed essentiallyas described previously (20). ELISA plates were coated with(per well) 200 ng of human sCD4 in 100 µl of PBS, pH 7.4,instead of carbonate/ bicarbonate buffer. Polyclonal Ig froman HIV-1-infected patient was used as a primary antibody insteadof polyclonal (rabbit) antisera. As a negative control, totalprotein from extracts of 293 cells transfected with empty vectorwas used. Competitive inhibition of binding with sCD4 was assessedby serial dilution of sCD4 (1:3, 1:9, 1:27) from 500 ng/wellcompared to no sCD4 in a 96-well plate that was previously coatedwith CD4, followed by incubation with either gp140 ${Delta}$ CFI or r-gp160(Intracel).

DNA injections in mice. Six-week-old, female BALB/c mice were injected intramuscularlywith 100 µg of purified plasmid DNA suspended in 200 µlof normal saline. For each plasmid DNA, a group of four micewas injected three times at intervals of 2 weeks. The mice werebled 2 weeks after the last injection, and sera were collectedand stored at 4°C.

Immunization of guinea pigs. Six-week-old, female Huntley guinea pigs were injected intramuscularlywith 500 µg of purified plasmid DNA encoding the CXCR4-tropicHXB2 gp145 ${Delta}$ CFI suspended in 400 µl of normal saline. Foreach plasmid DNA, a group of four guinea pigs was injected threetimes at intervals of 2 weeks. The guinea pigs were bled 2 weeksafter the last injection, and sera were collected and storedat 4°C. The guinea pigs received a boost with replication-defectiverecombinant adenovirus (ADV) expressing CXCR4-tropic HXB2 gp140 ${Delta}$ CFIas described previously (50, 54, 56) and were bled 2 weeks afterADV injection.

Quantitation of antibody response. Immunoprecipitation and Western blotting were used to detectthe antibodies that bind to native envelope proteins. Sera fromimmunized mice were used to immunoprecipitate gp160 from celllysates of gp160-transfected 293 cells and were compared withwell-characterized monoclonal antibodies 1726 and 1727 (NIHAIDS Research and Reference Reagent Program) against the V3loop of HIV-1 IIIb. Indicated dilutions were used to immunoprecipitategp160 from the cell lysate (400 µg). Immunocomplexes wereseparated by SDS-7.5% PAGE and analyzed by immunoblotting usingpolyclonal antibody against gp160.

Reactivity of gp140 ${Delta}$ CFI with monoclonal antibodies. The binding ability of several monoclonal antibodies—2F5,2G12, F105, and b12—to gp140 ${Delta}$ CFI was determined as above.Antibody (5 µg) was used to immunoprecipitate gp140 ${Delta}$ CFIfrom 100 µl of membrane-free supernatant from 293 cellstransfected with the expression vector expressing gp140 ${Delta}$ CFI.The same volume of supernatant from cells transfected with emptyvector was used as a control. Antibodies were obtained fromthe AIDS Research and Reference Reagent Program. The [³⁵S]methionine-labeledsupernatant was incubated with progressively diluted (5, 1,and 0.1 µg) monoclonal antibodies 2F5 and 2G12 along withHIV-1 IgG, and the intensity of the gp140 ${Delta}$ CFI band was determinedby quantitative phosphorimaging.

Sucrose density gradient centrifugation. The oligomerization of gp140 ${Delta}$ CFI was investigated by sucrosegradient centrifugation followed by Western blot analysis. Supernatantsfrom cells transfected with the gp140 ${Delta}$ CFI expression vector werecentrifuged at 16,000 x g at 4°C to remove debris. The supernatantswere further centrifuged at 100,000 x g to remove vesicles andmembrane fragments. The clarified supernatants were concentrated30-fold with a UFV2GC10 filter (Millipore). The concentratedsupernatants (750 µl) were loaded onto a 10%-to-40% continuoussucrose gradient and centrifuged in a Sorvall rotor (model TH641)for 20 h at 40,000 rpm at 4°C. Fractions of 1 ml were collectedmanually and analyzed by both nonreducing and reducing SDS-5%PAGE. Equal volumes from each fraction were used to immunoprecipitategp140 ${Delta}$ CFI by HIV-1 IgG, and the separated proteins were detectedby Western blotting with a rabbit polyclonal antibody to gp160.Cross-linked phosphorylase b (Sigma) was used as a high-molecular-weightmarker.

Molecular exclusion chromatography. Gel filtration using Superdex 200 gel filtration column (AmershamPharmacia, Piscataway, N.J.) was calibrated with markers withmolecular masses of 669, 440, 232, and 158 kDa individuallyand together. The proteins were chromatographed using membrane-freesupernatant containing gp140 ${Delta}$ CFI prepared as described for sucrosegradients. Proteins from relevant fractions were separated bySDS-10% PAGE and transferred to Immobilon-P membrane (Millipore).gp140 ${Delta}$ CFI was detected by immunoprecipitation followed by Westernblotting as described above, and band intensity was determinedby densitometry using the ImageQuant program. The molecularweight was determined from the curve of K_average (gel phasedistribution coefficient) versus log (molecular weight) of thestandards.

Analysis of CTL response. Spleens were removed aseptically, gently homogenized to a single-cellsuspension, washed, and resuspended to a final concentrationof 5 x 10⁷ cells/ml. Cells were incubated for 7 days in thepresence of interleukin 2 (10 U/ml) and either irradiated peptide-pulsedsplenocytes from naive mice or an irradiated stable cell lineexpressing the full-length gp160 BC-env/rev (18). Three typesof target cells were used: peptide-pulsed P815 cells (ATCC TIB64),BC10ME cells stably expressing gp160, and BC10ME cells (11)pulsed with peptides derived from gp160 sequence. Target cellswere labeled with ⁵¹Cr for 90 min and washed three times withRPMI 1640 medium with 10% fetal bovine serum, 2 mM glutamine,5 x 10^-5 M ß-mercaptoethanol and amphotericin (Fungizone)(250 U/ml), and resuspended in this medium. Cytolytic activitywas determined in triplicate samples using all different targetcell dilutions in a 5-h ⁵¹Cr release assay (34).

Neutralization assay. Antibody-mediated neutralization was assessed in MT-2 cellswith HIV-1 IIIB. Neutralizing antibody titers in the MT-2 assaywere defined as the reciprocal serum dilution at which 50% ofcells were protected from virus-induced killing as measuredby neutral red uptake (26). Assay stocks of HIV-1 IIIB wereproduced in H9 cells.

RESULTS

Top

Results

Development of HIV Env vectors. To develop Env glycoprotein variants that might better inducehumoral immunity after DNA immunization, a series of plasmidexpression vectors was generated (Fig. 1). Full-length HIV Env(gp160) was highly expressed in the absence of HIV accessoryproteins at levels $>=$ 10-fold higher than Rev-dependent viral gp160in transfected 293 cells as seen by Western blot analysis (Fig.2A and B), and the relevant mutant proteins were detected atthe expected apparent molecular weights (Fig. 2C to E). As mightbe anticipated, gp160 expressed from the synthetic gene wasnot efficiently processed in transfected 293 cells, similarto previously published studies (3, 57), presumably becausethe high levels of gp160 saturate the cellular proteases responsiblefor cleavage, as overexpression of furin can overcome this problem(3); nonetheless, processed gp160 was detected with a monoclonalantibody to gp41 (Fig. 2B, lanes 3 to 9). This result is consistentwith previous studies of viral gp160 in transfected cells, inwhich 75% may remain unprocessed (29).

Synthetic HIV gp160 induced toxicity in transfected cells, withcell rounding and detachment evident within 48 h (compare Fig.3A and B). This cytotoxicity was reduced by elimination of theCOOH-terminal cytoplasmic domain. Env protein that terminatedat amino acid 752 (gp150) was less cytotoxic than gp160, whilethe shorter proteins (gp145 and gp140) produced little or noeffect (Fig. 3C, D, and E, respectively).

View larger version (82K):
[in this window]
[in a new window]
FIG. 3. Cytotoxicity of full-length gp160 is eliminated by deletion of the COOH-terminal cytoplasmic domain. Cell rounding and detachment were not observed in control-transfected 293 cells (A), in contrast to full-length gp160 (B), and were observed to a lesser extent in cells transfected with gp150 (C), in contrast to gp145 (D) or gp140 (E).

To alter Env immunogenicity, two different approaches were explored.First the effects of glycosylation were evaluated. Two setsof mutations were introduced into both gp160 and gp150 (Fig.1). The first set included 11 potential N-linked glycosylationsites ( ${Delta}$ gly11), and the second set included an additional sixsites downstream ( ${Delta}$ gly17) that were eliminated by site-directedmutagenesis. Expression studies showed that the glycosylationmutants were efficiently expressed, and the glycosylation mutantprotein was appropriately reduced in size compared to wild-typegp160 or gp150, consistent with reduced N-linked glycosylation(Fig. 2D). These proteins were also readily detected on thecell surface by flow cytometry (data not shown). As noted below,these glycosylation mutants had little effect on the generationof antibodies to Env, so efforts were therefore directed toa different set of mutants.

The second approach involved a series of internal deletionsdesigned to stabilize and expose functional domains of the proteinthat might be present in an extended helical structure priorto the formation of the six-member coiled-coil structure inthe hairpin intermediate (10, 52). To generate this putativeprehairpin structure, the cleavage site was removed to preventthe proteolytic processing of the envelope and stabilize theprotein by linking it covalently to the gp41 extracellular and/ortransmembrane domain. To reduce toxicity and enhance stability,the fusion peptide domain was deleted. The heptad repeats inthe envelope protein are important tertiary structure domainsinvolved in trimer formation (23). The sequence between theheptad repeats was removed to stabilize the formation of trimersand eliminate formation of the hairpin intermediate. These ${Delta}$ CFIdeletions were introduced into full-length gp160 and COOH-terminaltruncation mutants. Cells transfected with vectors encodinggp140 ${Delta}$ CFI, gp145 ${Delta}$ CFI, and gp160 readily expressed these proteins(Fig. 2E). gp140 ${Delta}$ CFI, which lacks the transmembrane domain, waseasily detected in the supernatant (Fig. 4), indicating thatit can give rise to soluble antigen. Some gp160 was also notedin media from cells transfected with wild-type gp160, likelyreflecting some membrane contamination of the supernatant fractionor release of gp160 after cell lysis due to toxicity inducedby gp160.

View larger version (47K):
[in this window]
[in a new window]
FIG. 4. Expression of soluble ${Delta}$ CFI HIV-1 envelope variants. Results of immunoprecipitation and Western blot analysis of supernatants from the indicated transfected cells are shown.

The antigenic structure of gp140 ${Delta}$ CFI was analyzed using differentconformation-dependent monoclonal antibodies and by biochemicalanalysis. At least four well-defined monoclonal antibodies—2F5,2G12, F105, and b12—bound specifically to gp140 ${Delta}$ CFI (Fig.5A). The specificity of binding by 2F5 and 2G12, in comparisonto anti-HIV-1 IgG, was confirmed further by performing a quantitativedose-response analysis (Fig. 5B). Similarly, gp140 ${Delta}$ CFI retainedits ability to interact specifically with its natural receptor,CD4, and its binding was competitively inhibited by sCD4 (Fig.5C). As a negative control, total protein from 293 cells transfectedwith empty vector was used, in which the optical density was $<=$ 0.1, confirming the specificity of binding with this assay.Finally, the ability of gp140 ${Delta}$ CFI to oligomerize was tested biochemically.By sucrose density gradient analysis and Western blot analysis,gp140 ${Delta}$ CFI predominantly formed trimeric and dimeric oligomers(Fig. 5D). The patterns that have been observed are typicalof other envelope proteins, with monomers, dimers, trimers,and multimers. Notably, there was a significant component oftrimer in the equilibrium gradient, showing that the proteinis capable of forming the appropriate oligomer. It is also likelythat some aggregation occurs, and additional high-molecular-mass(>500-kDa) multimers were observed in fraction 1 (data notshown), typical of such analyses (55). The proteins in all fractionsmigrated at the position of the monomer under denatured reducingconditions (Fig. 5D, lower panel). Membrane-free supernatantswere analyzed by molecular exclusion chromatography using Superdex200. The major peak was found at the calculated size of $~$ 315kDa (Fig. 5E), consistent with the predicted size of the trimericcomplex. Flow cytometric analysis of cell surface envelope glycoproteinsindicated that both gp140 ${Delta}$ CFI and gp145 ${Delta}$ CFI were synthesized andprocessed within cells. Both proteins were expressed on thecell surface after transfection, as recognized by flow cytometrywith HIV Ig or two different monoclonal antibodies, 2F5 and2G12 (Fig. 6). Though gp140 ${Delta}$ CFI was secreted from cells, it wasalso readily detected on the cell surface, likely due to itshigh level of synthesis in transfected cells, as has been describedfor other secreted and highly expressed proteins previously(24, 45).

View larger version (44K):
[in this window]
[in a new window]
FIG. 5. Interaction of gp140 ${Delta}$ CFI with defined monoclonal antibodies or CD4 and biochemical analysis for oligomerization of gp140 ${Delta}$ CFI. (A) Analysis of the antigenic structure of soluble gp140 ${Delta}$ CFI with monoclonal antibodies. The envelope glycoproteins from the supernatants of the 293 cells transfected with the vector expressing gp140 ${Delta}$ CFI were immunoprecipitated with either 5 µg of monoclonal antibodies 2F5, 2G12, F105, and b12 or with 5 µg of HIV-1 Ig. The proteins were analyzed by SDS-PAGE and detected by Western blotting using the polyclonal antibodies against gp160. The arrow indicates the position of gp140 ${Delta}$ CFI. A nonspecific (ns) band that cross-reacted with the antibody is indicated. (B) Quantification of 2F5 and 2G12 binding to gp140 ${Delta}$ CFI using the indicated concentrations of each antibody as shown compared to a nonreactive Ig isotype (control). The intensity of the gp140 ${Delta}$ CFI band was determined by quantitative phosphorimaging. The arrow indicates the position of gp140 ${Delta}$ CFI. (C) Interaction of soluble gp140 ${Delta}$ CFI protein with CD4. Binding of gp140 ${Delta}$ CFI and gp160, compared to that of controls transfected with vector alone, in an ELISA with CD4 is shown (left panel). The values represent the mean and standard deviation (error bars) for each point. The ability of sCD4 to compete with binding to these envelopes is shown (right panel). (D) Biochemical analysis of soluble gp140 ${Delta}$ CFI oligomerization. Western blot analysis to detect the gp140 ${Delta}$ CFI in different fractions, fractions 1 to 10, after sucrose density gradient. Fraction 1 represents the greatest density, and fraction 10 represents the least density. Equal volumes of the samples from each fraction were analyzed in an SDS-polyacrylamide gel under nonreducing conditions, except the molecular weight marker. The proteins were detected by Western blotting using polyclonal antibody against gp160. The positions of dimer, trimer, and aggregates are shown. The lower panel shows the presence of monomer in these fractions run under reducing conditions. (E) Molecular-exclusion chromatography of soluble of gp140 ${Delta}$ CFI. Membrane-free supernatant containing gp140 ${Delta}$ CFI was analyzed on a Superdex 200 column and compared with a mixture of molecular weight standards; the position of each marker is indicated by arrows. Fractions were analyzed for gp140 ${Delta}$ CFI by immunoprecipitation followed by Western blotting and were quantitated by densitometry.

View larger version (25K):
[in this window]
[in a new window]
FIG. 6. Confirmation of cell surface expression of gp140 ${Delta}$ CFI and gp145 ${Delta}$ CFI by flow cytometry. 293 cells were transfected with either a control vector or vectors expressing gp140 ${Delta}$ CFI or gp145 ${Delta}$ CFI, as indicated. The transfection efficiency ( $~$ 90%) was calculated based on staining after transfection of the same number of cells under identical conditions with an equal amount of DNA expressing ß-galactosidase. Cell surface expression was detected by FACS analysis using Ig purified from the sera of HIV-1-infected individuals (HIV Ig) or different monoclonal antibodies, 2F5 and 2G12. The transfected cells are labeled at the top, and the different antibodies are indicated at right. Light dotted lines indicate FACS analysis with control human Ig, and dark dotted lines indicate FACS analysis with human monoclonal antibodies against HIV-1.

Increased antibody response after DNA vaccination with ${Delta}$ CFI Envmutants. The ability of these Env proteins to elicit a humoralimmune response was determined in mice by injection with theseplasmid DNA expression vectors. To enhance the sensitivity ofthese sera for native conformational epitopes, antibody responseswere monitored by their ability to immunoprecipitate wild-typegp160 from cell lysates followed by SDS-PAGE and Western blotting(Fig. 7). In some cases, antibody reactivity was also confirmedby indirect immunofluorescence (data not shown). To quantitatethe antibody response, immunoprecipitation with different dilutionsof immunized mouse sera was performed. The intensity of thegp160 band was determined by densitometry and standardized relativeto a positive control serum used to normalize data between experiments.The approximately linear dose response of gp160 intensity withserum dilution allowed quantification of the anti-gp160 antibodyresponse in mouse serum. Data from immunized mice showed thatnone of the wild-type Env proteins, neither the gp160, gp150,gp145, nor gp140 COOH-terminal truncations, generated consistentlyhigh antibody responses (Fig. 7A). Of these proteins, wild-typegp140 was somewhat more effective than gp160 in generating antibodyresponses, but these responses remained low and inconsistent.Immunization with vectors designed to express glycosylation-deficientenvelope proteins also did not improve the humoral response(data not shown). In contrast, the ${Delta}$ CFI mutants substantiallyincreased the anti-gp160 antibody response when the transmembranedomain was truncated (Fig. 7A [gp145, gp140, and gp128 versusgp160 and gp150]). gp140 ${Delta}$ CFI provided more-consistent and greaterincreases in antibody response than gp128 ${Delta}$ CFI (Fig. 7B). In allcases, these vectors that encoded gp140 ${Delta}$ CFI, but not those thatencoded wild-type gp140, induced antibodies that were reactiveto native gp160 (Fig. 7C).

View larger version (29K):
[in this window]
[in a new window]
FIG. 7. Antibody response against HIV-1 envelope proteins in DNA immunized mice. (A) Comparison of the antibody response in mice immunized with gp140 ${Delta}$ CFI or other Env plasmid expression vectors. Sera were collected 2 weeks after the last immunization and used to immunoprecipitate codon-altered gp160 from lysates of transfected 293 cells as described before. The quantitation of the immunoprecipitated gp160 was done as described for panel B. The average of the normalized data has been presented as a bar diagram. Error bars are indicated. (B) Antibody responses in mice immunized with gp140 ${Delta}$ CFI or gp128 ${Delta}$ CFI relative to a V3-specific monoclonal antibody standard (monoclonal antibody 1727). Antisera from immunized mice were diluted in immunoprecipitation buffer, and 1 µl of each diluted serum was used to immunoprecipitate codon-altered HIV-1 gp160 from lysates of transfected 293 cells as described in the legend to Fig. 3A. The gels were scanned, and the intensity of the gp160 band was determined by densitometry using the program ImageQuant and presented relative to the intensity of gp160 immunoprecipitated with positive control sera (rabbit anti-gp160), which was used to normalize data between experiments. These data are presented graphically to facilitate comparison among groups. Monoclonal antibody 1727 interacts with the V3 loop of HIV IIIb and was kindly provided by the NIH AIDS Research and Reference Reagent Program, from Jon Laman. (C) Antibody responses in mice immunized with gp140 or gp140 ${Delta}$ CFI were determined by immunoprecipitation and Western blotting. Animals received two booster doses (100 µg) of the same plasmid, 2 weeks apart. Sera (1 µl) collected 2 weeks after the last immunization were used to immunoprecipitate codon-optimized HIV-1 gp160 from lysates of transfected 293 cells containing 400 µg of total protein. Each lane corresponds to the serum from an animal immunized with either the control vector (lanes 1 and 2), CXCR4-tropic gp140 (lanes 3 to 6), or plasmid that expresses gp140 with the indicated mutant functional domains (lanes 7 to 10). A mouse monoclonal antibody to gp160 (HIV-1 V3 monoclonal [IIIB-V3-13]; NIH AIDS Research and Reference Reagent Program) was used as a positive control (lane 11).

To determine whether these modifications of Env adversely affectedCTL responses, spleen cells from immunized mice were testedfor their ability to lyse relevant target cells. All mice immunizedwith wild-type codon-altered Env vectors, including COOH-terminaldeletion mutations (Fig. 8A) or glycosylation mutants (Fig.8B) elicited strong CTL responses against cell lines pulsedwith HIV Env peptides. These findings were confirmed using stablytransfected cells that expressed Env (data not shown). Importantly,gp140 ${Delta}$ CFI, which elicited increased antibody responses relativeto the comparable wild-type Env, readily induced CTL responsesto native Env, as did other ${Delta}$ CFI mutants (Fig. 8C). Additionof anti-CD8 antibody inhibited cytolytic activity, as did depletionof CD8 cells using magnetic beads coupled with anti-CD8 antibody,thus confirming a CTL response to these immunogens after geneticimmunization (data not shown). This response was detectablefor at least 6 months after immunization. Finally, the abilityof gp140 ${Delta}$ CFI to generate neutralizing antibodies was tested todetermine whether this immunogen can elicit a neutralizing antibodyresponse to functional Env on virus. This experiment was performedwith guinea pigs due to the high background and variabilityin mouse antisera in the neutralization assay. Because theseguinea pigs do not respond as efficiently to plasmid DNA alone,a DNA prime-ADV boost protocol was used, as previously describedin an Ebola virus challenge model (50, 54). Four animals wereimmunized with gp140 ${Delta}$ CFI plasmid DNA as was done for mice andreceived boosters at week 6 with 10¹¹ particles of ADV-gp140 ${Delta}$ CFI.Titers were increased from undetectable levels (<1:20) inpreimmune animals to $>=$ 1:100 after immunization (Fig. 8D) (P =0.02), documenting that the antisera react with functional Envon virus.

View larger version (23K):
[in this window]
[in a new window]
FIG. 8. CTL response against HIV-1 envelope proteins in DNA-immunized mice and generation of a neutralizing antibody response in guinea pigs. (A) The CTL response to CXCR4-tropic Env and indicated deletion mutants is shown. (B and C) The CTL responses to CXCR4-tropic envelope with glycosylation site and ${Delta}$ CFI mutations are shown, respectively. Spleen cells were isolated from immunized mice 2 weeks after the final immunization and stimulated in vitro with irradiated cells expressing gp160 with addition of human interleukin 2 (5 U/ml) at day 4. The cytolytic activity of the restimulated spleen cells was tested after 7 days against V3 peptide-pulsed BC10ME cells. Similar findings were observed with target cells that stably express full-length Env (data not shown). (D) Preimmune sera were collected from four guinea pigs prior to immunization or after DNA priming and ADV boosting with gp140 ${Delta}$ CFI as described in the text. Both preimmune sera and postimmune sera were diluted, and neutralizing activity was measured by reduction of HIV-IIIB virus compared to the untreated control. Neutralizing antibody titers were analyzed as previously described (26). The data represent the dilutions at which the sera can neutralize the virus in the MT2 assay. Standard deviations are indicated.

DISCUSSION

Top

Discussion

To develop DNA vaccine candidates for HIV, we developed a seriesof synthetic genes designed to express HIV Env mutants in humancells. In the absence of HIV regulatory proteins, these codon-alteredenvelope protein genes were readily expressed in human cells.Like other DNA vaccines, immunization with these vectors elicitedstrong CTL responses in mice, and antibody responses were notrobust in mice immunized with wild-type Env expression vectors.Mutations in highly conserved N-linked glycosylation sites didnot significantly alter humoral or cellular immune responseto native Env. In contrast, a mutant Env with deletions in thecleavage site, fusion domain, and a region between the heptadrepeats elicited a more-potent humoral immune response and retainedits ability to stimulate Env-specific CTL.

The immune response to HIV infection in long-term nonprogressors(9, 35) and HIV-exposed sex workers suggests that specific viralimmunity may limit infection and the symptoms of disease, butno single characteristic correlates with protective immunity.Depletion of CTLs in chronically infected macaques enhancesviremia (19, 46). In humans, higher CTL responses correlatewith lower viral load and stabilization of clinical symptoms(30, 33). In animal models, passive transfer of neutralizingantibodies can also contribute to protection against virus challenge(8, 15, 28, 31, 32, 36, 43, 48). Neutralizing antibody responsescan also be developed in HIV-infected individuals (8, 36, 43)and are associated with lower viral loads in long-term nonprogressors(25). Though broadly neutralizing antibody responses are uncommon,they are directed largely against the Env protein of the virus(40, 49). In early human vaccine trials, gp120 protein immunogenshave yielded disappointing results: vaccine-induced antibodieshave not been broadly neutralizing and have sometimes enhancedinfection in vitro (5, 7, 12, 17, 51). Monomeric gp120 losesoligomer-dependent epitopes and does not include sequences inthe ectodomain of the gp41 that become exposed during virusentry (6, 27, 32). It is assumed that broadly neutralizing antibodiesmust bind well to native gp120/gp41 on the surface of the virus(8).

Recent reports suggest that gp160 forms trimers in vivo andthe domain required for trimer formation resides in the ectodomainof the gp41 (55). Such trimeric forms of HIV envelope proteinare likely to present different epitopes to the immune systemcompared to monomeric gp120. In addition to the linear epitopesin the envelope, this trimeric structure is likely to displayconformational epitopes important for B-cell triggering of arelevant antibody response. In this regard, gp140 ${Delta}$ CFI, whichinduced the greatest antibody response, is released in a solubletrimeric form (Fig. 4 and 5). This finding was confirmed bothby sedimentation gradient and gel filtration analysis (Fig.5D and E). Biochemical purification is likely to yield dimericand monomeric forms, and another potential advantage of gene-basedvaccines is the ability to make physiologically relevant complexesin vivo. In contrast to gp140 ${Delta}$ CFI, wild-type Env did not elicithigh-titer antibody responses. The toxicity of Env in mammaliancells has been seen previously and could limit both the amountand duration of envelope protein expression in vivo, thus affectingimmunogenicity. The envelope is also heavily glycosylated, andremoval of partial or complete gp120 glycosylation sites hasresulted in higher titers of strain-specific neutralizing antibodyresponses to mutant simian immunodeficiency viruses in monkeys,presumably through enhanced exposure of critical epitopes (4,39). Though it seemed reasonable that deglycosylation wouldreveal epitopes otherwise masked in the native protein, we didnot observe enhanced immune reactivity by DNA vaccination usingdifferent glycosylation site mutants, both in gp160 and gp150.This difference with the previous study is likely due to thefact that DNA vaccination rather than viral infection was utilizedfor immunization. Though glycosylation mutants are unlikelyto prove helpful with the former method of immunization, itremains possible that modification of glycosylation sites maybe effective with other vectors or adjuvants.

HIV-1 Env is proteolytically cleaved by a cellular proproteinconvertase into gp120 and gp41. The gp41 subunit is composedof cytoplasmic, transmembrane, and ectodomain segments. Therole of the ectodomain of the envelope in membrane fusion, particularlyits hydrophobic glycine-rich fusion peptide, is well established.Two regions with heptad coiled-coil repeats in the ectodomainof gp41 are involved in viral fusion (13, 53). Upon fusion,these two alpha-helices, connected via a disulfide-stabilizedloop (14, 21, 44), presumably undergo a transient conformationalchange to a fusion active state. These changes allow the formationof a six-member helical hairpin intermediate structure thatpresumably exposes the fusion peptide at the NH₂ terminus ofgp41, allowing fusion to the target cell membrane (2, 22). The ${Delta}$ CFI mutation was intended to eliminate cleavage of gp140, removethe unstable hydrophobic region, and stabilize oligomer formation.Though detailed structural data are not yet available on thisprotein, the immunologic and biochemical analyses indicatedthat these mutations retain native antigenic determinants, definedby known monoclonal antibodies, and oligomeric properties, similarto native viral envelope (Fig. 5). The gp140 ${Delta}$ CFI mutant alsoelicits both humoral and cellular immune responses, probablyby virtue of protein stabilization and secretion. For example,the neutralizing epitope (ELDKWAS) in the ectodomain of gp41(31) is present in the series of deletions and truncations ofthe envelope, and gp140 ${Delta}$ CFI is reactive with the 2F5 neutralizingmonoclonal antibody that binds to this epitope (Fig. 6). Itis also relevant that protein misfolding is unlikely to accountfor enhanced antibody responses, as several vaccine candidatesthat induced such responses showed expected cell surface expressionand/or secretion (Fig. 4, 6, and 7 [gp140 ${Delta}$ CFI and gp145 ${Delta}$ CFI]),and several immunogens that were not appropriately exposed hadno effect (data not shown).

Importantly, these immunogens also induced CTL responses toEnv. It is evident that DNA immunization often elicits predominanthumoral or cellular immune responses that are determined byfeatures of the immunogen that are not understood. For example,we have found that responses to Env are primarily cellular whilethose to Nef display enhanced humoral immunity. Though gp128 ${Delta}$ CFIinduced slightly more potent CTL activity, gp140 ${Delta}$ CFI was betterable to elicit an antibody response, retained the 2F5 epitope,and was readily able to elicit such responses, both to peptide-pulsedcells and stably transduced target cells. Thus, the enhancedhumoral immune response introduced by this vaccine candidatedid not appear to diminish the CTL response. Taken together,these results suggest that gp140 ${Delta}$ CFI serves as an improved immunogenthat can more effectively elicit a neutralizing antibody responseagainst the envelope by DNA vaccination while preserving itsability to induce a CTL response.

ACKNOWLEDGMENTS

We thank Nancy Barrett for preparation of the figures, MousumiPaul and Judith Stein for advice, Nancy Sullivan for discussionsand thoughtful suggestions, and Cherilyn Davis and Ati Tislericsfor manuscript preparation. We thank Janet Hoff of the Universityof Michigan for assistance during the immunization of the animals.The following reagents were kindly provided through the AIDSResearch and Reference Reagent Program: HIV-1 V3 monoclonalantibody (IIIB-V3-01 and IIIB-V3-13) from Jon Laman, b12 fromDennis Burton and Carlos Barbas, 2G12 and 2F5 from Hermann Katinger,and F105 from Marshall Posner. We thank Richard Wyatt and PeterD. Kwong for helpful discussion on analyzing the trimeric structureof the protein and Shazad Majeed for assistance in the fast-performanceliquid chromatography analysis.

FOOTNOTES

* Corresponding author. Mailing address: Vaccine Research Center, NIAID, National Institutes of Health, 40 Convent Dr., Bethesda, MD 20892-3005. Phone: (301) 496-1852. Fax: (301) 480-0274. E-mail: gnabel{at}mail.nih.gov.

REFERENCES

Top