Inoculation of guinea pigs with YU2 gp120 and gp140(−/GCN4) proteins in selected adjuvants.
Guinea pigs, four animals per group (animal 47 in group B died during the course of the study), were inoculated with either monomeric YU2 gp120 or trimeric YU2 gp140(−/GCN4) protein immunogens emulsified in Ribi or the GSK adjuvants, AS01B, AS02A, or AS03. Animals were inoculated a total of five times, with 4- to 7-week intervals between inoculations. Test bleeds were collected 10 days after each inoculation, and the isolated sera were subjected to ELISA and HIV-1 neutralization assays.
ELISA analysis of the antibodies in the sera elicited by gp120 and gp140(−/GCN4).
After the fourth inoculation, sera were collected and tested for binding activity to YU2gp120 proteins by ELISA. All groups generated high titers of anti-gp120 IgG titers, with the end-point titers ranging from 3.2 × 105
to 7.5 × 10 6
(Table ), which is consistent with the results from our previous studies in mice or rabbits (22
). Although most animals generated relatively high antibody titers, the animals from group A (monomeric gp120 in Ribi adjuvant) and both groups of animals inoculated with either monomer or trimers in the AS03 adjuvant had slightly lower end-point binding titers (Table ). Both the AS01B and AS02A adjuvants stimulated approximately fivefold higher titers of binding antibodies than Ribi adjuvant in most animals inoculated with either monomeric or trimeric glycoproteins (Table ).
Envelope glycoprotein and V1, V2, and V3 variable loop reactivity of the sera from immunized guinea pigsa
Because much of the neutralizing response elicited by YU2 gp120 was directed against the V1 region (see below), we sought to determine if these elicited binding antibodies were predominantly elicited against the V1/V2 variable loops. We first compared the antibody binding titer against gp120 and a gp120 with the V1/V2 loop deleted (gp120ΔV1V2). We observed that for the 15 gp120-inoculated animals, 7 exhibited a fivefold decrease in recognition of gp120ΔV1V2 compared to recognition of wild-type gp120 (Table , animals 41, 42, 44, 45, 52, 54, and 55 of groups A, B, and C). In the group D animals (gp120 in the AS03 adjuvant), which had lower titers to gp120, there was not an observable binding decrease to gp120ΔV1/V2 proteins. In the animals inoculated with YU2 gp140(−/GCN4) proteins, 5 out of 16 animals displayed a fivefold decrease in binding titer to the gp120ΔV1/V2 proteins compared to wild-type gp120 (Table ). These data suggest that although there are significant levels of binding antibodies directed toward the V1/V2 region, it is not the single dominant antibody response elicited by either gp120 or gp140 glycoproteins (Table ).
Serum ELISA binding activity to V1, V2, and V3 peptides.
As shown below, much of the neutralizing activity elicited by monomeric YU2 gp120 can be inhibited by a single YU2 V1 peptide but much less so by the YU2 V3 peptide; we sought to determine how much of the overall binding antibody repertoire was directed against these variable loop regions. By ELISA, we tested for the serum binding activity to the pool of V1/V2 peptides, to the single 15-mer V1 a02 peptide, and to the V3 peptide (Table and data not shown). Most of the animal sera showed high binding end-point titers for the V1 a02 peptide, ranging from 3.13 × 105 to 1.5 × 10 6; the exceptions were that all the group A animals and animals 59 and 61 had lower binding titers to this peptide (Table ). All the animal sera demonstrated similar binding patterns to the V1/V2 peptide pool, suggesting that most of the binding antibodies are directed toward the V1 a02 peptide. The binding titers of most of the sera to the V3 peptide were reasonably high (1.25 × 10 4 to 1.5 × 10 6) but were less than the levels obtained for binding to the V1 peptides (Table ). Taken together, these data suggested that both YU2 gp120 and YU2 gp140(−/GCN4) proteins elicited significant levels of V1- and V3-loop-directed binding antibodies.
Trimeric gp140(−/GCN4) proteins elicit more potent and broader neutralization than monomeric gp120 proteins.
After the fourth injection, sera were collected and assayed for neutralization activity in a titration format against the homologous YU2 virus and the relatively sensitive virus SF162 using a FACS-based PBMC neutralization assay. We selected sera from animals from groups D and H that had been inoculated with monomeric YU2 gp120 or trimeric YU2 gp140(−/GCN4) proteins in the same AS03 adjuvant. Not all groups were analyzed to conserve limited sera for subsequent analyses. Sera were tested at an initial dilution of 1:10 and serial fivefold dilutions. For YU2, at the initial 1:10 serum dilution, the trimeric YU2 gp140(−/GCN4) animal sera had a mean (± standard error of mean) neutralization activity of 89% ± 8.0%, while monomeric YU2 gp120 animal sera displayed an average neutralization activity of 56% ± 20% (Fig. ). The IC50 values for the reciprocal dilutions of the sera elicited by the gp140 glycoproteins were at least fivefold higher, ranging from 37 to 186, than the values for monomeric YU2 gp120-elicited sera, which ranged from 6 to 32. Similarly, for SF162, the sera from YU2 gp140(−/GCN4) protein-injected animals (group H) displayed approximately fivefold higher IC50 values than the YU2 gp120 protein-injected animals (group D) (Fig. ).
FIG. 1. Titration of neutralization activity in sera after four inoculations of YU2 gp120 (group D) or gp140(−/GCN4) (group H) with the GSK AS03 adjuvant. Sera were tested for neutralization of YU2 (A) and SF162 (B) at decreasing serum concentrations. (more ...)
To assess the neutralization breadth of the sera elicited in selected adjuvants, we tested immune sera obtained after both the third and fourth inoculations of protein-adjuvant. Sera were assayed at a 1:5 dilution against the homologous YU2 virus and several heterologous viruses, as shown in Fig. . Within the gp120-adjuvant groups, the percent neutralization of the YU2 and SF162 isolates generally increased from the third to the fourth inoculation (Fig. ). After the third inoculation, sporadic neutralization of IIIB and 89.6 was detected, but this diminished following the fourth inoculation. Similarly, there were only two serum samples that possessed greater than 50% neutralization of the Bx08 and BR07 isolates. In general, sera isolated from the animals inoculated with gp140(−/GCN4) in the GSK adjuvants displayed more frequent and more potent neutralization of the YU2, IIIB, and 89.6 isolates than was achieved by the gp120-inoculated animals (Fig. ). From the sera isolated after the third inoculation compared to after the fourth, increased neutralization of the YU2 and SF162 isolates was observed, whereas IIIB neutralization diminished slightly and 89.6 neutralization diminished dramatically. Sporadic, low-level neutralization of the BR07 and Bx08 isolates was observed in the gp140-GSK adjuvant groups, with the YU2 gp140(−/GCN4)-AS02A group exhibiting the most neutralization breadth, albeit of relatively modest potency (Fig. ).
FIG. 2. PBMC, FACS-based neutralization assay against five HIV-1 primary isolates and one T-cell-line-adapted isolate, IIIB. Shown are values from a single-round in vitro neutralization with guinea pig sera after the third and fourth inoculations at a 1:5 dilution (more ...) Assessment of sera neutralization breadth with a recently standardized HIV-1 assay.
We sought to examine the breadth of neutralization following a fifth inoculation of the gp120 and gp140(−/GCN4) proteins in the recently standardized assay designed to permit the cross comparison of neutralization elicited by diverse immunogens (33
). Due to the analysis described in the present report, there were no sera remaining from the third and fourth protein inoculations; therefore, we compared the sera following the second inoculation to that following the fifth. Initially, we tested the ability of the sera to neutralize a panel of clade B Env-pseudotyped viruses at a single dilution of 1:5. As seen in Fig. , the sera from the gp140(−/GCN4) protein inoculated in the GSK adjuvants efficiently neutralized YU2, BaL, JR-CSF, and JR-FL deleted of the 301 glycan (JR-FLΔ301 [30
]). In general, and as before, the sera derived from YU2 gp140(−/GCN4) emulsified in the GSK adjuvants displayed more potent neutralizing activity than did the sera elicited by gp120 in the GSK adjuvants. The difference in neutralization between the gp140- and gp120-elicited sera was statistically significant as determined by a nonparametric Mann-Whitney test. For most viruses, the P
value derived by this analysis ranges from 0.0001 to 0.056 (see Fig S1 in the supplemental material).
FIG. 3. (A) Neutralization activity against eight pseudotyped HIV primary isolates. Shown are values from a single-round in vitro neutralization with guinea pig sera after the second and fifth inoculations at a 1:5 dilution against the indicated isolates. Numbers (more ...)
By comparing the serum neutralization potency from samples after inoculations 2 and 5, we observed a general increase in homologous neutralization and, when achieved, heterologous neutralization generally increased (Fig. ). Interestingly, in contrast to this trend, the YU2 gp140(−/GCN4)-elicited sera after inoculation 2 (and after inoculation 3 in the previous analysis) neutralized the 89.6 isolate; however, most of this activity declined following inoculations 4 and 5. After inoculation 2, all sera elicited by the YU2 gp140(−/GCN4) in AS02A adjuvant (group G) neutralized the 89.6 virus at values greater than 80% (Fig. ). We assessed the potency of these sera by serial dilution and 89.6 neutralization to derive IC50 values in excess of 1:640 for the individual serum (see Fig. ). In general, the sera in group G (YU2 gp140 in AS02A) displayed the greatest potency, as most individual animal serum samples from this group could neutralize 7 of the 10 isolates tested at the level of 50% or greater (Fig. ). As before, we performed a titration of the neutralizing activity against the homologous YU2 virus from sera elicited in the matched AS03 adjuvant; the gp140(−/GCN4)-inoculated animals displayed significantly higher neutralization titers with average IC50 values 15-fold higher than the gp120-inoculated animals (1:12 for the monomer compared to 1:186 for the trimer) (Fig. ). Consistent with the homologous neutralization titers, when the heterologous virus JR-CSF was tested in the titration assay, a moderate threefold higher IC50 value was observed for the gp140-GCN4-inoculated animals compared to that for the gp120-inoculated animals (data not shown). In summary, in both the PBMC-based assay and the standardized Env-pseudotyped virus neutralization assay, we confirmed that trimeric gp140(−/GCN4) proteins elicit more potent and modestly broader neutralization than the monomeric gp120 proteins.
FIG. 6. Peptide neutralization specificity mapping of selected sera against heterologous primary isolates. (A) Percent neutralization of 89.6 is shown in a bar graph format of individual sera elicited by the gp140(−/GCN4) proteins at the indicated serial (more ...) The adjuvants AS01B, AS02A, and AS03 elicit more potent and broader neutralizing antibodies compared to the matched immunogen in Ribi adjuvant.
By inspection of the neutralization data presented in Fig. and , it is apparent that especially with the gp140(−/GCN4) immunogens, the GSK family of adjuvants is superior to the commercially available Ribi adjuvant. After inoculation 4 (Fig. ), the sera from three out of four animals inoculated with YU2 gp120 in Ribi adjuvant could not neutralize either the homologous isolate YU2 or the lab-adapted isolate IIIB or the sensitive primary isolate SF162 (Fig. ). From this group, only animal 42 had relatively potent neutralizing activity against the YU2 and SF162 isolates. The sera from most animals in all groups injected with monomeric gp120 in the GSK series of adjuvants had more potent neutralization activity against YU2 and SF162 but less neutralization activity against IIIB. The 2 animals in group B (AS01B adjuvant) were the exception and did neutralize IIIB in the 60 to 70% range at the 1:5 dilution values tested (Fig. ).
When the mean neutralizing values elicited by the gp140 proteins in each of the four adjuvants were statistically compared by a Mann-Whitney nonparametric analysis, it was apparent that the GSK adjuvants all elicited higher neutralizing activity at the single dilution point analyzed (Fig. ). For animals inoculated with the trimeric gp140(−/GCN4) proteins, the percentage of homologous YU2 neutralization ranged from 91 to 99% in the AS01B, AS02A, and AS03 adjuvants and ranged from 0 to 68% in Ribi adjuvant group (P < 0.03) (Fig. and ). For the lab-adapted isolate IIIB and the sensitive primary isolate SF162, the percent neutralization values ranged from 50 to 93% in the AS01B, AS02A, and AS03 adjuvants, while it was below 50% in the Ribi adjuvant (group E) (P < 0.03) (Fig. and ). In summary, the GSK family of adjuvants improves the potency of the neutralizing antibody response with either the monomeric or trimeric YU2 envelope glycoprotein immunogens compared to the commercially available Ribi adjuvant.
FIG. 4. Comparison of gp140-elicited neutralization of the YU2 (A), SF162 (B), and IIIB (C) viruses in the four adjuvants. The horizontal bars indicate the mean neutralization value of each adjuvant group at a 1:5 serum dilution. For each virus, the Ribi adjuvant (more ...) Mapping homologous YU2 neutralization specificity elicited by monomeric and trimeric immunogens using peptide inhibition of neutralization.
To define the neutralization specificity elicited by monomeric gp120, we performed peptide inhibition assays. Initially we focused on the V3 region and utilized an assay that we had established previously (22
). We used the anti-V3 loop monoclonal antibodies 447-D and 2442 to validate the assay, as previously described (22
). Briefly, animal sera were preincubated with synthesized peptides derived from the YU2 V3 region. In sera, these peptides could potentially form a complex with the V3 loop-directed antibodies and thus block the neutralization activity against V3-specific neutralization determinants. The percent inhibition of neutralization was obtained by comparing the percent neutralization in the presence or absence of peptide to identify V3-directed neutralization present in a given serum sample.
For the V3 analysis, serum samples remaining in greatest abundance after the previous analysis were selected. Ten serum samples from the YU2 gp120-inoculated animals were analyzed in this experiment. As shown in Table , the presence of V3 peptide in the assay did not greatly inhibit the homologous YU2 neutralization activity for most of the sera (30% or less). The exception was the serum from animal 54 in the GSK AS02A adjuvant group, which showed a 47% activity decrease following incubation with the V3 peptide. However, many of the gp120-elicited sera that possessed relatively weak neutralization also displayed some minor fluctuations in neutralization activity with and without the V3 peptide. Because of the increased variability in the assay, and by inspection of the robust V1 peptide inhibition data below, we interpret the results to indicate that the homologous neutralization activity elicited by YU2 gp120 was not predominantly against the V3 loop.
Neutralization of YU2 virus and V1, V2, and V3 loop peptide adsorptions of the sera elicited by YU2 gp120 and gp140(−/GCN4)
When we performed similar V3 peptide inhibition assays with the more potent YU2 gp140(−/GCN4)-elicited sera, the data clearly demonstrated that little homologous neutralizing activity was V3 directed (Table ). The percent inhibition of neutralization mediated by the V3 peptide was less than 10% for 11 of 12 animals, and the sera from animals 61 and 63 displayed V3-mediated inhibition levels of 24% and 17% (Table ). These data were consistent with our previous rabbit immunogenicity, study which demonstrated that the homologous neutralization activity elicited by trimeric YU2 gp140(−/GCN4) protein was not predominantly directed toward V3 loop.
We then attempted to examine if the specific neutralization activity was focused on the V1 or V2 loops. To develop a peptide inhibition assay for this gp120 region, we first selected two serum samples (animals 44 and 46 from group B) and preincubated each of the sera with a pool of overlapping 15-mer peptides derived from the YU2 V1 and V2 loops (Fig. ) (see Materials and Methods). Somewhat surprisingly, we found that the V1/V2 peptide pool inhibited most of the neutralization activity of these two serum samples. For animal 46, 100% of the neutralization activity was inhibited by the V1/V2 peptide pool (Fig. and Table ), and for animal 44, 61% of neutralization was inhibited (Table ). These results suggested that monomeric YU2 gp120 elicits neutralizing antibodies predominantly focused on V1 and/or V2 loops. To better define the specificity, we divided the V1 and V2 peptides into three test groups, the first containing two peptides, designated V1V2 pool 1. The second group contained a single peptide V1 a02 that we designated pool 2 (we had found that a similar peptide derived from HXBc2 inhibited HXBc2 homologous neutralization; J. Mascola and G. Nabel, unpublished observations). A pool of the four most-C-terminal V1/V2 peptides was designated V1V2 pool 3 (Table and Fig. ). We then tested the inhibition effects of these three peptide sets on 10 selected animal serum samples. We found that the single peptide V1 a02, derived from the C terminus of V1 loop, inhibited homologous neutralization of 7 of 10 serum samples at the 100% level, 2 of 10 serum samples at the 70% level, and 1 serum sample at the 40% level (Table and Fig. ). These results strongly suggested that the neutralization activity against the homologous isolate YU2 elicited by monomeric gp120 glycoprotein was focused to a single epitope within the V1 loop. Consistent with this observation, most of the other peptides within the V1 and V2 loop did not show significant inhibition of neutralization. The one exception was that the serum from animal 48 was inhibited 35% by the V1V2 pool 1 and 47% by the V1V2 pool 3 (Table ). However, this particular serum sample displayed diverse effects, and the data were difficult to interpret. By definition, the inhibition of neutralization should not exceed the 100% level, which was already achieved by the V1 a02 peptide itself.
To examine if the YU2 gp140(−/GCN4) elicited neutralization activity toward V1 and V2 loops, we preincubated the sera with the three V1/V2 peptide groups. As shown in Table , 11 of 12 animal serum samples had less than 20% activity inhibition by both V1V2 pool 1 and pool 3. Compared to peptide pools 1 and 3, there was more inhibition of neutralization inhibited by the peptide a02. Seven of 12 samples had less than 50% inhibition of neutralization by V1 peptide a02, while 5 of 12 samples displayed 50 to 83% inhibition. These data suggested that trimeric YU2 gp140(−/GCN4) elicited neutralization activity against the autologous virus YU2 that was partially, but not predominantly, directed toward the V1 and V2 loops, in contrast with neutralizing specificity elicited by monomeric YU2 gp120. In a similarly designed assay, we also analyzed selected sera elicited by the gp140(−/GCN4) glycoproteins for inhibition by preincubation with a 2F5-binding peptide. No reduction in homologous neutralization activity by the 2F5-binding peptide could be observed, although this peptide could efficiently inhibit 2F5-mediated neutralization in the same experiment (data not shown).
Mapping heterologous neutralization specificity by peptide inhibition.
We observed that although homologous YU2 neutralization and heterologous neutralization of several isolates increased with repeated inoculations, neutralization of 89.6 declined after inoculations 2 through 5 (see Fig. and ). Less dramatically, neutralization of the IIIB isolate also declined slightly after inoculations 3 to 4 (Fig. ). To determine where the initial 89.6 neutralizing activity was directed, we performed adsorptions of the sera using the YU2 V1/V2 peptide pools and with the V3 peptide prior to performing neutralization assays. As shown in Fig. , when we performed the adsorption/neutralizations over a range of serum dilutions from 1:10 to 1:640, most of the neutralization could be removed by the V3 peptide (in this case, a 40% reduction in neutralization is roughly a threefold difference in viral entry), but the V1/V2 peptides did not affect neutralization. From these data, IC50 values for 89.6 neutralization could be determined and were greater than 1:640 for animals 68 to 72. Given that we could now detect some V3-directed heterologous neutralization, we analyzed selected and relatively potent sera against other selected isolates. In contrast to 89.6, heterologous neutralization of JR-CSF elicited by either the trimer or the monomer in ASO2A adjuvant following the fifth inoculation was not V3 directed (Fig. ). We also mapped selected sera against BaL.01 following the fifth inoculation and found that similar to 89.6, neutralization was mostly adsorbed by the YU2 V3 peptide (approximately 10-fold reduction of viral neutralization) (see Fig S3 in the supplemental material), whereas most JR-FLΔ301 neutralization following the fifth inoculation was not V3 directed (data not shown).