Adjuvant-dependent innate and adaptive immune signatures of risk of SIVmac251 acquisition

Abstract

A recombinant vaccine containing Aventis Pasteur's canarypox vector (ALVAC)-HIV and gp120 alum decreased the risk of HIV acquisition in the RV144 vaccine trial. The substitution of alum with the more immunogenic MF59 adjuvant is under consideration for the next efficacy human trial. We found here that an ALVAC-simian immunodeficiency virus (SIV) and gp120 alum (ALVAC-SIV + gp120) equivalent vaccine, but not an ALVAC-SIV + gp120 MF59 vaccine, was efficacious in delaying the onset of SIVmac251 in rhesus macaques, despite the higher immunogenicity of the latter adjuvant. Vaccine efficacy was associated with alum-induced, but not with MF59-induced, envelope (Env)-dependent mucosal innate lymphoid cells (ILCs) that produce interleukin (IL)-17, as well as with mucosal IgG to the gp120 variable region 2 (V2) and the expression of 12 genes, ten of which are part of the RAS pathway. The association between RAS activation and vaccine efficacy was also observed in an independent efficacious SIV-vaccine approach. Whether RAS activation, mucosal ILCs and antibodies to V2 are also important hallmarks of HIV-vaccine efficacy in humans will require further studies.

Figure 1

Study design and vaccine efficacy. Unless stated otherwise, in all the figures, the data for the alum group are in red (n = 27) and those for the MF59 group (n = 27) and the controls (n = 24) are in blue and black, respectively. (a) Study design. Animals were immunized with ALVAC (vCP2432) expressing SIV genes gag-pro and gp120TM, derived from the founder variant of SIV_mac251 designated M766r and boosted with the SIV_mac251 M766 gp120-gD (referred to in the text as gp120) and SIV_smE660 gp120-gD CG7V (referred to in the text as gp120) proteins formulated in alum (200 mg) or MF59 (100 mg). (b,c) SIV_mac251 acquisition. The number of intrarectal (IR) exposures before viral acquisition was assessed in the alum (b) and MF59 (c) groups, relative to controls, using the log–rank test of the discrete-time proportional hazards model. (d,e) SIV RNA levels in the plasma. Error bars show logarithmic mean ± s.d. of animals vaccinated with alum (d) or MF59 (e), and all historical and concurrent control animals (n = 47). (f) SIV DNA copies in rectal mucosa at 2 weeks after infection (horizontal lines, median) in all vaccinated and concurrent controls (n = 23). (g) Number of viral variants in the plasma at 2–4 weeks after infection in all groups. (h) Percentage of CD4⁺ T cell changes in the blood over time (weeks) (mean ± s.e.m.).

Figure 2

Env-specific IgG responses in mucosa. Unless differently stated, data throughout are in red for the alum group (n = 27), blue for the MF59 group (n = 27) and black for the controls (n = 24). (a,b) Specific activity of rectal IgG-binding antibodies to the gp130 of SIV_mac251 (a) and the gp140 of SIV_smE660 (b). (c,d) Specific activity of IgG-binding antibodies to the gp70-V1/V2 scaffold of SIV_mac251 (c) or SIV_smE660 (d) in rectal secretion. (e–g) Neutralizing antibodies to Tier 1 SIV_mac251.6 (e), phagocytosis index (f) and ADCC (g). (h,i) Gp120-specific complement activation (h) and V2-specific phagocytosis (i). Each symbol represents one animal, and horizontal lines represent the medians. The Mann–Whitney–Wilcoxon test was used to compare continuous factors between two groups.

Figure 3

Serum and rectal IgG to cyclic V2, PB homing markers and SIV_mac251 acquisition. (a–d) IgG to cV2 of SIV_mac251 (MF59, n = 26; alum, n = 20; controls, n = 12) (a,c) and SIV_sme543.3 (MF59 and alum, n = 26; controls n = 18) (b,d) in sera (a,b) and rectal mucosa (c,d). Titers or response units are shown. Horizontal lines are medians. (e,f) SIV_mac251 acquisition in vaccinated animals that had postvaccination rectal IgG response units to cV2 of SIV_smE543 >0 (solid lines) or 0 (dotted lines) in the alum group (e) and in the MF59 group (f). Although IgG responses to cV2 peptides in the mucosa were generally low, and high background was noted in some animals in the pre-vaccination samples, postvaccination data were supported by an independent multiplex assay of mucosal V2 responses (Supplementary Fig. 3). (g) Phenotypic characterization of PBs. Plots represent the gating strategy, where gating is represented by squares or circles and the arrows represent the parent-to-daughter population flow for each line. (h,j) Frequency of vaccine-induced α4β7⁺ PBs (h) or vaccine-induced CXCR3⁺ (j) in macaques at day 7 after the final immunization (alum, n = 22; MF59, n = 23. (i) Frequency of vaccine-induced α4β7⁺ PBs in the blood of 17 humans enrolled in the RV132 and RV135 HIV-vaccine trials. Blood was collected at 14 d from vaccination (Wilcoxon signed–rank test).

Figure 4

Vaccine-induced changes in the frequency and function of mucosal ILC subsets. (a) Representative flow cytometric plots defining ILCs in the rectal mucosa of rhesus macaques. ILCs were identified using a side-scatter versus forward-scatter gate and phenotypically defined as CD3⁻CD20⁻ and NKG2A⁺ or NKp44⁺ cells, or as NKG2A⁻NKp44⁻cells. Purple line defines the gates used to calculate CD20⁻CD3⁻ cells and NKp44⁺NKG2A⁺ cells. (b,c) Relative frequency of NKp44⁺ (b) and NKG2A⁻NKp44⁻ (c) cells at week 25 in the MF59-vaccinated (n = 26) and alum-vaccinated animals (n = 27) and unimmunized controls (n = 30). Horizontal lines represent the medians. (d) Frequency of NKp44⁺ cells producing IL-17 after Env-stimulation (alum, n = 24; MF59, n = 22). (e) Kinetic of the recruitment of NKp44⁺ ILCs that produce IL-17 after in vitro stimulation in six immunized animals (mean values ± s.d.). (f) Correlation between the percentage of mucosal IFN-γ⁺NKG2A⁻NKp44⁻ cells and time of acquisition in the alum group. (g) The ability of individual sets of aggregate data to predict protection in the MF59 and alum groups was compared among animals that were infected in four or fewer challenges (≤4) or after five or more challenges (≥5), using logistic regression and leave-one-out cross-validation (Supplementary Fig. 5). The best model predicting alum-conferred protection from SIV acquisition was obtained when features identified as “cells” were used (accuracy, >81%). The features selected in the “cells” regression model are presented as a bar plot (y axis). The length and direction of each bar is proportional to the relative predictive contribution of each feature in the model (x axis, regression coefficient). Features associated with a high risk of SIV acquisition are in blue; those associated with low risk are in red.

Figure 5

Transcription and immune profiles associated with the risk of SIV_mac251 acquisition. (a) Heat map (left) showing the expression of a 12-gene signature predicting protection by the alum group. The 12 genes that compose this signature were identified using the prevax samples and tested on the postvax samples (post-first and post-third) (right). (b) Receiver-operating characteristic curve presenting the accuracies of the 12-gene classifier on the postvax samples. (c) Network inference based on the 12 genes included in the predictive signature. The list of 12 genes was uploaded in GeneMania. Edges are based on co-localization, co-expression and canonical interaction (pathways). Genes associated with a high risk of SIV acquisition in the alum-treated macaques are presented as blue nodes, and genes associated with a low risk of SIV acquisition are presented as red nodes. Gray nodes represent genes associated with the RAS pathway that did not significantly correlate with SIV acquisition. (d) Integrative analysis between the transcriptomic data, the humoral markers and the glycan modifications associated with acquisition. Least-square regression using, as independent variables, the pre-vaccination expression of the 12-gene signature identified to be predictive of protection by the alum-adjuvanted vaccine (c), and as dependent variables, the humoral markers of protection (Supplementary Table 3) was performed using the function spls of the R package mixOmics. The network presents all the pairs of features that are significantly positively correlated with each other (absolute Pearson correlation, R > 0.223; P < 0.05).

Figure 6

RAS-related genes associated with protection conferred by the SIV gp96 + gp120 alum vaccine. Heat-map representation of the expression of the 37 genes associated with protection by ALVAC–SIV + gp120 alum (24-h post-third vaccination) and SIV-gp96 + gp120 (1 week post-third vaccination). Genes that were positively correlated with the number of SIV challenges to infection after ALVAC–SIV + gp120 alum vaccination significantly overlapped with genes associated with the number of SIV challenges to infection after vaccination with SIV-gp96 + gp120 (Supplementary Table 7). Leading-edge analysis revealed that 37 genes were contributing to the enrichment. (a) Heat map of the expression of the 37 genes after vaccination with ALVAC–SIV + gp120 alum. The expression intensities are represented using a blue-white-red color scale. Rows correspond to transcripts, and columns correspond to profiled samples. Samples were ordered by increasing expression of the 37 genes. (b) Heat map of the expression of the 37 genes after SIV-gp96 + gp120 vaccination. (c) Network inference based on the 37 genes associated with protection conferred by the ALVAC–SIV + gp120 alum and SIV-gp96 + gp120 vaccines. List of 37 genes was uploaded in GeneMANIA to test their interaction with NRAS. The principal hub of network, NRAS, was placed in the center of the network, whereas genes directly associated with the RAS pathway were placed in the inner circle, and genes indirectly associated with RAS were placed in the outer circle. Edges correspond to interaction identify by GeneMANIA colored by the type of interaction between genes (co-expression, co-localization or canonical pathway interaction). Nodes represent genes associated with a low risk of SIV_mac251 acquisition (red) and intermediary genes identified by GeneMANIA (gray).