A first-in-human germline-targeting HIV nanoparticle vaccine induced broad and publicly targeted helper T cell responses

Abstract

The engineered outer domain germline targeting version 8 (eOD-GT8) 60-mer nanoparticle was designed to prime VRC01-class HIV-specific B cells that would need to be matured, through additional heterologous immunizations, into B cells that are able to produce broadly neutralizing antibodies. CD4 T cell help will be critical for the development of such high-affinity neutralizing antibody responses. Thus, we assessed the induction and epitope specificities of the vaccine-specific T cells from the IAVI G001 phase 1 clinical trial that tested immunization with eOD-GT8 60-mer adjuvanted with AS01_B. Robust polyfunctional CD4 T cells specific for eOD-GT8 and the lumazine synthase (LumSyn) component of eOD-GT8 60-mer were induced after two vaccinations with either the 20- or 100-microgram dose. Antigen-specific CD4 T helper responses to eOD-GT8 and LumSyn were observed in 84 and 93% of vaccine recipients, respectively. CD4 helper T cell epitope "hotspots" preferentially targeted across participants were identified within both the eOD-GT8 and LumSyn proteins. CD4 T cell responses specific to one of these three LumSyn epitope hotspots were observed in 85% of vaccine recipients. Last, we found that induction of vaccine-specific peripheral CD4 T cells correlated with expansion of eOD-GT8-specific memory B cells. Our findings demonstrate strong human CD4 T cell responses to an HIV vaccine candidate priming immunogen and identify immunodominant CD4 T cell epitopes that might improve human immune responses either to heterologous boost immunogens after this prime vaccination or to other human vaccine immunogens.

Fig. 1.. Vaccine-specific CD4 and CD8 T cell responses target eOD-GT8 and LumSyn.

(A) Shown is a schematic of the T cell analyses in the IAVI G001 clinical trial. Cryopreserved PBMCs obtained by leukapheresis 2 weeks after second immunization (week 10) were analyzed by ICS assay. Tfh cells in LN fine needle aspirates (FNA) 3 weeks after first and second vaccination were measured by flow cytometry (weeks 3 and 11, respectively; fig. S2). (B) Boxplots show the interquartile range of background-adjusted frequencies of cytokine-positive eOD-GT8 and LumSyn-specific CD4 T cells at week 10. Statistically positive responses were determined by MIMOSA and are indicated by colored closed circles. Nonresponders are indicated by open gray triangles. The number of positive responders of the participants tested and the percentage of positive responders are indicated above each graph. (C) Frequencies of vaccine-specific cTfh-like cells were measured in the PBMCs at week 10 as the background-adjusted percentage of IL-2⁺ or CD40L⁺ CXCR5⁺ CD4 T cells of total circulating CD4 T cells after peptide stimulation. No positivity call was applied to the vaccine-specific cTfh cells. Individual data points are indicated as colored open circles by dose group. (D) Shown are background-adjusted frequencies of cytokine-positive CD8 T cells in response to eOD-GT8 and LumSyn in the PBMC samples collected at week 10. Positive responses (by MIMOSA) are indicated by colored closed circles, and nonresponders are indicated by open gray triangles. Response rates are indicated above each graph. Significant differences between the placebo and 20- and 100-μg dose groups were determined by Barnard’s test (two-sided, α = 0.05).

Fig. 2.. Antigen-specific CD4 T cells have diverse phenotypes by K-means clustering analysis.

K-means clustering was conducted on CD4 T cells that were positive for at least two cytokines or activation markers in the eOD-GT8, LumSyn, negative control, and CMV stimulations by ICS assay. (A) Heatmap indicates proportion of cells that were positive for each marker within the specified cluster. GzB, granzyme B. (B) Shown are the background-adjusted percentages of positive CD4 T cells for each cluster of the total CD4 T cells stimulated with CMV pp65 (top), eOD-GT8 (middle), or LumSyn (bottom). Box plots indicate the median and interquartile range.

Fig. 3.. Immunodominant T cell responses to eOD-GT8 and LumSyn identified by epitope mapping by ICS.

(A) CD4 T cell responses to eOD-GT8 and corresponding subpools (13 to 14 peptides each), eOD-GT8-1, eOD-GT8-2, and eOD-GT8-3, are shown. Frequencies of cytokine positive T cells are background-adjusted. The numbers of positive responders out of participants tested and percent of positive responders are indicated above each graph. Positive responses were determined by Fisher’s exact test and are indicated by colored closed circles. Negative responses are indicated by open gray triangles. Box plots indicate the median and interquartile range. Significant differences between the placebo and 20- and 100-μg dose groups were determined by Barnard’s test (two-sided, α = 0.05). ns, not significant. (B) Shown are individual peptide-specific CD4 and CD8 T cell responses to LumSyn (41 peptides; 1 to 175 amino acids) and eOD-GT8-3 by ICS assay. Amino acid numbering is in relative to the eOD-GT8–LumSyn linear sequence. Each row (y-axis tick) indicates a participant. Statistically significant responses are plotted using one horizontal line representing the amino acids covered by the given peptide. Nonresponders were included as blank rows. Because of the overlapping design of the peptides, recognition of a single epitope may be indicated by overlapping peptide responses. (C and D) Shown is the percentage of vaccine recipients, by dose group, with positive CD4 T cell responses to the eOD-GT8-3 (C) and LumSyn (D) individual peptides (numbered sequentially in order from N terminus to C terminus relative to the eOD-GT8–LumSyn linear sequence), hotspots, and combinations of hotspots.

Fig. 4.. Associations of the frequencies of T and B cell responses within and between peripheral and LN compartments.

Frequencies of eOD-GT8⁺ IgG memory B cells and vaccine-specific CD4 T and cTfh (CD40L⁺ CXCR5⁺) CD4 T cells were evaluated in PBMCs collected at 2 weeks after second vaccination. Frequencies of eOD-GT8⁺ IgG GC B cells, total GC B cells, and total GC Tfh cells were analyzed in draining LN FNA samples collected 3 weeks after second vaccination. The direction and degree of correlation by Spearman r is indicated by color and size of the interior square. *P < 0.05, **P < 0.01, and ***P < 0.001, by active vaccine treatment arm: 20-μg, 100-μg, and combined dose groups.