Interaction dynamics between innate and adaptive immune cells responding to SARS-CoV-2 vaccination in non-human primates

As SARS-CoV-2 variants continue evolving, testing updated vaccines in non-human primates remains important for guiding human clinical practice. To date, such studies have focused on antibody titers and antigen-specific B and T cell frequencies. Here, we extend our understanding by integrating innate and adaptive immune responses to mRNA-1273 vaccination in rhesus macaques. We sorted innate immune cells from a pre-vaccine time point, as well as innate immune cells and antigen-specific peripheral B and T cells two weeks after each of two vaccine doses and used single-cell sequencing to assess the transcriptomes and adaptive immune receptors of each cell. We show that a subset of S-specific T cells expresses cytokines critical for activating innate responses, with a concomitant increase in CCR5-expressing intermediate monocytes and a shift of natural killer cells to a more cytotoxic phenotype. The second vaccine dose, administered 4 weeks after the first, elicits an increase in circulating germinal center-like B cells 2 weeks later, which are more clonally expanded and enriched for epitopes in the receptor binding domain. Both doses stimulate inflammatory response genes associated with elevated antibody production. Overall, we provide a comprehensive picture of bidirectional signaling between innate and adaptive components of the immune system and suggest potential mechanisms for the enhanced response to secondary exposure.


Fig. S3. Additional analysis of heavy chain somatic hypermutation (SHM). (A)
Same data as shown in Fig. 2B, divided by animal.N=604 cells at week 2 and 1,393 cells at week 6.92 cells from animal 16C303 at week 2 are not included, as no matching week 6 data were available from that animal.Distributions were compared using a 2sided unpaired Wilcoxon test.P = 0.0025 for 16C222 and 0.019 for 34941.(B) Heavy chain SHM down-sampled to one cell per lineage to account for possible confounding effects due to correlations in SHM among cells in the same lineage.N=641 lineages at week 2 and 1,238 lineages at week 6.Distributions were compared using a 2-sided unpaired Wilcoxon test, P = 3.8E-4.(C) The same data as in (B), divided by animal, except that 87 lineages from animal 16C303 at week 2 are not included.N=554 lineages at week 2 and 1,238 lineages at week 6.Distributions were com-pared using a 2-sided unpaired Wilcoxon test.P = 1.1E-5 for 16C235 and 7.5E-4 for 34941.IgK and IgL sequences, respectively, at week 6.Distributions were compared using a 2-sided unpaired Wilcoxon test, P = 0.0402 and 0.0087 for kappa and lambda, respectively.(B) The same data as in (A), divided by animal.92 cells from animal 16C303 at week 2 are not included, as no matching week 6 data were available from that animal.P = 0.028, 2.5E-4, and 0.044 for 16C283-lambda, 34941-kappa, and 36186-lambda, respectively.(C) Kappa and lambda chain SHM down-sampled to one cell per lineage to account for possible confounding effects due to correlations in SHM among cells in the same lineage.N=311 IgK and 330 IgL lineages at week 2 and 531 IgK and 707 IgL lineages at week 6.Distributions were compared using a 2-sided unpaired Wilcoxon test, P = 0.016 and 0.003 for kappa and lambda, respectively.(D) The same data as in (C), divided by animal, except that 43 IgK and 44 IgL lineages from animal 16C303 at week 2 are not included.N=268 IgK and 286 IgL lineages at week 2 and 531 IgK and 707 IgL lineages at week 6.Distributions were compared using a 2-sided unpaired Wilcoxon test.P = 6.4E-6, 0.028, 2.5E-4, and 0.044 for 16C235-lambda, 16C283-lambda, 34941-kappa, and 36186-lambda, respectively.

Fig. S1 .
Fig. S1.Study design and sort strategies.(A) Vaccination schedule and timeline of sample collection for sort of each cell type.(B) Sorting strategy for B cells (pink gates) explored on Fig 1 and innate cells (green gates) explored on Fig 6. (C) Sorting strategy for non-specific T cells (memory CD4 T cells from DMSO control) and antigen-specific T cells explored on Fig 4.
Fig. S2.B cell clonal dynamics of 6 animals.Alluvial plots, with grey lines representing singleton B cells and colored lines represent expanded lineages.Lines that start or end at zero, including all singleton, indicate B cell lineages that were observed in only a single time point.Colored lines that span the graph represent lineages found at both time points.The thickness of each line at either side of the panel is proportional to the number of cells in that lineage at the corresponding time point, with the cumulative number of cells in all lineages indicated on the y-axis.A total of 521 cells from week 2 and 1,241 cells from week 6 were analyzed.No plot is shown for animal 16C303, for which no week 6 IG sequences are available.
Fig. S4.Additional analysis of CDR H3 length.(A)Same data as shown in Fig.2C, divided by animal.N=554 lineages at week 2 and 1,238 lineages at week 6.87 lineages from animal 16C303 at week 2 are not included, as no matching week 6 data were available from that animal.Distributions were compared using a 2-sided unpaired Wilcoxon test.P = 0.01, 0.012, and 0.0048 for 16C222, 34941, and 36186, respectively.(B) CDR H3 length distribu-tions calculated using all cells in the data set.Although CDR H3 length is effectively constant within a lineage and thus not statistically independent, this shows the full post-antigen selection repertoire, including any clonal expan-sion.N=696 cells at week 2 and 1,393 cells at week 6.Distributions were compared using a 2sided unpaired Wilcoxon test, P = 1.2E-7.(C) The same data as in (B), divided by animal, except that 92 cells from animal 16C303 at week 2 are not included.N=604 cells at week 2 and 1,393 cells at week 6.Distributions were compared using a 2-sided unpaired Wilcoxon test.P = 0.029, 5.6E-5, and 0.0059 for 16C222, 34941, and 36186, respectively.

Fig. S5 .
Fig. S5.Mutational burden of IG light chains.(A) Kappa and lambda light chain somatic hypermutation (SHM) of antigen-specific memory B cells.N=330 and 366 IgK and IgL sequences, respectively, at week 2 and 588 and 805IgK and IgL sequences, respectively, at week 6.Distributions were compared using a 2-sided unpaired Wilcoxon test, P = 0.0402 and 0.0087 for kappa and lambda, respectively.(B) The same data as in (A), divided by animal.92 cells from animal 16C303 at week 2 are not included, as no matching week 6 data were available from that animal.P = 0.028, 2.5E-4, and 0.044 for 16C283-lambda, 34941-kappa, and 36186-lambda, respectively.(C) Kappa and lambda chain SHM down-sampled to one cell per lineage to account for possible confounding effects due to correlations in SHM among cells in the same lineage.N=311 IgK and 330 IgL lineages at week 2 and 531 IgK and 707 IgL lineages at week 6.Distributions were compared using a 2-sided unpaired Wilcoxon test, P = 0.016 and 0.003 for kappa and lambda, respectively.(D) The same data as in (C), divided by animal, except that 43 IgK and 44 IgL lineages from animal 16C303 at week 2 are not included.N=268 IgK and 286 IgL lineages at week 2 and 531 IgK and 707 IgL lineages at week 6.Distributions were compared using a 2-sided unpaired Wilcoxon test.P = 6.4E-6, 0.028, 2.5E-4, and 0.044 for 16C235-lambda, 16C283-lambda, 34941-kappa, and 36186-lambda, respectively.
CDR H3 length distribu-tions calculated using all cells in the data set.Although CDR H3 length is effectively constant within a lineage and thus not statistically independent, this shows the full post-antigen selection repertoire, including any clonal expan-sion.N=696 cells at week 2 and 1,393 cells at week 6.Distributions were compared using a 2sided unpaired Wilcoxon test, P = 1.2E-7.(C) The same data as in (B), divided by animal, except that 92 cells from animal 16C303 at week 2 are not included.N=604 cells at week 2 and 1,393 cells at week 6.
2C, divided by animal.N=554 lineages at week 2 and 1,238 lineages at week 6.87 lineages from animal 16C303 at week 2 are not included, as no matching week 6 data were available from that animal.Distributions were compared using a 2-sided unpaired Wilcoxon test.P = 0.01, 0.012, and 0.0048 for 16C222, 34941, and 36186, respectively.(B)