Envelope-Specific Recognition Patterns of HIV Vaccine-Induced IgG Antibodies Are Linked to Immunogen Structure and Sequence

Abstract

Background: A better understanding of the parameters influencing vaccine-induced IgG recognition of individual antigenic regions and their variants within the HIV Envelope protein (Env) can help to improve design of preventive HIV vaccines. Methods: Env-specific IgG responses were mapped in samples of the UKHVC003 Standard Group (UK003SG, n = 11 from UK) and TaMoVac01 (TMV01, n = 17 from Tanzania) HIV vaccine trials. Both trials consisted of three immunizations with DNA, followed by two boosts with recombinant Modified Vaccinia Virus Ankara (MVA), either mediating secretion of gp120 (UK003SG) or the presentation of cell membrane bound gp150 envelopes (TMV01) from infected cells, and an additional two boosts with 5 μg of CN54gp140 protein adjuvanted with glucopyranosyl lipid adjuvant (GLA). Env immunogen sequences in UK003SG were solely based on the clade C isolate CN54, whereas in TMV01 these were based on clades A, C, B, and CRF01AE. The peptide microarray included 8 globally representative Env sequences, CN54gp140 and the MVA-encoded Env immunogens from both trials, as well as additional peptide variants for hot spots of immune recognition. Results: After the second MVA boost, UK003SG vaccinees almost exclusively targeted linear, non-glycosylated antigenic regions located in the inter-gp120 interface. In contrast, TMV01 recipients most strongly targeted the V2 region and an immunodominant region in gp41. The V3 region was frequently targeted in both trials, with a higher recognition magnitude for diverse antigenic variants observed in the UK003SG (p < 0.0001). After boosting with CN54gp140/GLA, the overall response magnitude increased with a more comparable recognition pattern of antigenic regions and variants between the two trials. Recognition of most immunodominant regions within gp120 remained significantly stronger in UK003SG, whereas V2-region recognition was not boosted in either group. Conclusions: IgG recognition of linear antigenic Env regions differed between the two trials particularly after the second MVA boost. Structural features of the MVA-encoded immunogens, such as secreted, monomeric gp120 vs. membrane-anchored, functional gp150, and differences in prime-boost immunogen sequence variability most probably contributed to these differences. Prime-boosting with multivalent Env immunogens during TMV01 did not improve variant cross-recognition of immunodominant peptide variants in the V3 region.

Keywords: HIV; envelope-specific antibodies; epitope variant recognition; immunogen sequence; immunogen structure; vaccine.

Figure 1

Vaccination schedules in the UKHVC003 Standard Group and TaMoVac01 trial. The UKHVC 003 SG vaccine comprised DNA plasmids (8 mg/immunization) and MVA (10⁸ TCID50/immunization) coding for matched subtype C-derived CN54 based Env and ZM96 Gag, Pol, and Nef immunogen sequences. Of note, MVA-C expressed only the gp120 portion of Env. The CN54rgp140 protein (100 ug/immunization) was adjuvanted with 5ug GLA-AF and administered as two additional boosts. The TaMoVac I vaccination regimen included DNA vaccination (600 or 1,000 μg/immunization) with 7 plasmids (encoding for Env subtypes A, B, and C and Rev subtype B as well as Gag subtypes A, B, and RTmut subtype B) delivered intradermal, followed by MVA-CMDR (10⁸ pfu/immunization), expressing HIV gp150 (subtype E), Gag and Pol (subtype A), and two additional boosts with 100 μg of CN54rgp140 protein adjuvanted with 5 μg GLA-AF.

Figure 2

Peptide array design to cover recently transmitted global HIV strains. (A) Phylogenetic analysis of acute infection sequences (n = 350) from the Los Alamos database used for the selection of the eight isolates (in red) to design the peptide array. Purple diamonds indicate the isolates of the vaccine immunogens CMDR and CN54. Evolutionary analyses were conducted in MEGA6 (24) and the evolutionary history was inferred using the Neighbor-Joining method (25). The percentage of replicate trees (Bootstrap test 100 replicates) in which the associated taxa clustered together is shown above the branches (26). (B) Number of 15-mer amino acid (aa) sequences represented in the peptide array. The peptide array was aligned to the HxB2 sequence that was scanned with a sliding window of 11 aa using a three aa step. The Y-axis indicates the number of 11-mer aa sequences found in the array corresponding to a given 11-mer HxB2 window. (C) Peptide recognition amongst the known Los Alamos database HIV-1 sequences. 2,078 peptides produced 471,597 complete homology hits amongst 10,956 virus isolates. The Y-axis indicates the number of hits per peptide position, each subtype represented by a different color.

Figure 3

Mapping of antigenic regions targeted by Env-specific IgG responses in UKHVC003 and TaMoVac01. The frequency of responders (FOR, y-axis) to antigenic regions within the Envelope protein (A) after the second MVA (first row) and after CN54rgp140/GLA boosts (second row) is shown for the UKHVC_003 standard group (left panels, n = 11), and for TaMoVac01 recipients (right panels, n = 17). Immunodominant regions 1–8 are indicated. For region-specific IgG responses occurring in at least 25% of vaccinees, the mean fluorescence intensity (MFI) is shown in (B) after MVA (third row) and after CN54rgp140/GLA boosts (fourth row) for the two groups. IgG responses against individual antigenic regions were considered positive, if the corresponding fluorescence intensity was above 2,500 after subtraction of the pre-vaccination value. Mapping results from UKHVC003 were partially published previously (17).

Figure 4

Comparison of IgG responses targeting selected frequently recognized regions in UK003SG or TMV01. Shown is for the eight selected immunodominant regions [IDR1-8 (A–H)] for each analyzed vaccinee the mean recognition MFI over all tested antigenic variants at baseline, after the second boost with MVA and after the second boost with CN54gp140/GLA. The first HxB aa position of the 15 mer peptide and number of tested variants are indicated for each antigenic region. The mean MFI of responses for each antigenic region recognized within gp120 by above 25% of vaccinees are shown in (I). The results of the statistical analyses by Mann-Whitey test comparing data from the two trials are indicated only for (I). Data points from UK003SG and TMV01 are shown in magenta and light blue, respectively. The statistical analyses for differences in mean recognition magnitude for (A–H) is provided in Table 1.

Figure 5

Alignment of immunogen sequences for the eight selected immunodominant antigenic regions for immunogens included in TMV01 and UK003SG. From top to bottom; the first row indicates the Immunodominant region, HxB2 location and Region name and shows a representative sequence for this region. Row 2–4 show three sequence variants of the TMV01-DNA encoded Env proteins representing subtypes A, B, and C, respectively. Row 5 shows the MVA-CMDR-gp150 encoded subtype E sequence that was used during TMV01. Row 6 shows the CN54 sequence, which matches the DNA and MVA-C-gp120 encoded Env immunogens of UK003SG and the CN54gp140 recombinant protein administered in both trials.

Figure 6

Mapping of immunodominant peptide regions (IDR) to the structure of Env. (A) Color-coded solvent accessible surface area (SASA) of the eight IDRs projected on a native like Env trimer (C-clade consensus structure, PDB ID 8ck9). Glycans were removed and IDRs are highlighted in all protomers. Both IDR5.1 and IDR5.2 are combined as IDR5. (B) Eight IDRs projected onto the surface of a gp120-gp41 protomer from (A). (C) Location of the IDRs in the context of a CD4-induced (CD4i) gp120 structure (PDB ID 3jwd). Variable loops 1–4 which are deleted or not resolved in the structure are symbolized by dotted lines.

Figure 7

Phylogenetic heat map analyses of IgG recognition of the Env variable region 3 in UK003SG and TMV01 vaccine recipients. Shown is a heat map of mean signal intensities of IgG recognition of 36 peptide sequence variants of the variable region 3 tip (HxB304_ RKSIRIGPGSTFYAT) in the context of their phylogenetic relationship and their frequency of occurrence in the HIV database represented by the icon size. (A,B) Show the results after the second MVA boost, (C,D) for the results after the second CN54gp140/GLA boost with the vaccine. A maximum likelihood phylogenetic tree for 36 antigenic variants of the HIV V3 Tip region was generated after sequence of 75 amino acids was added as scaffolding to each of the 15 mer peptides represented on the peptide array. Please note the different heat map scale in (B). The arrows point to the CN54 isolate sequence in the respective trees.