High-throughput profiling of anti-glycan humoral responses to SIV vaccination and challenge

Abstract

Recent progress toward an HIV vaccine highlights both the potential of vaccines to end the AIDS pandemic and the need to boost efficacy by incorporating additional vaccine strategies. Although many aspects of the immune response can contribute to vaccine efficacy, the key factors have not been defined fully yet. A particular area that may yield new insights is anti-glycan immune responses, such as those against the glycan shield that HIV uses to evade the immune system. In this study, we used glycan microarray technology to evaluate anti-glycan antibody responses induced by SIV vaccination and infection in a non-human primate model of HIV infection. This comprehensive profiling of circulating anti-glycan antibodies found changes in anti-glycan antibody levels after both vaccination with the Ad5hr-SIV vaccine and SIV infection. Notably, SIV infection produced generalized declines in anti-glycan IgM antibodies in a number of animals. Additionally, some infected animals generated antibodies to the Tn antigen, which is a cryptic tumor-associated antigen exposed by premature termination of O-linked glycans; however, the Ad5hr-SIV vaccine did not induce anti-Tn IgG antibodies. Overall, this study demonstrates the potential contributions that glycan microarrays can make for HIV vaccine development.

Figure 1. Immunization schedule for the Ad5hr-SIV trial.

Each of the macaques underwent the same schedule. Ad5hr-SIV recombinant immunogens were administered at weeks 0 and 12 followed by envelope boosts at weeks 24 and 36. Intrarectal SIV_mac251 challenge occurred at week 42. Blood serum and plasma samples were collected at weeks 0, 38, 46, and 64.

Figure 2. Comparison of anti-glycan antibody profiles in macaques and humans.

Heat map showing glycan binding of circulating IgG (A) and IgM (B) in pre-vaccinated macaques (n = 38) and healthy humans (n = 30). Columns correspond to individual glycans organized into glycan families (see legend). Rows represent individual humans and macaques, which have been sorted by hierarchical clustering. Macaques and humans have highly similar repertoires of anti-glycan antibodies. Highlighted glycans are (1) 2′FucLac, (2) Hya8, (3) P1, (4) Ovalbumin, (5) LeA, (6) alpha-gal, (7) Forssman di, (8) Ac-Tn(Thr)-G-21, and (9) Ac-Tn(Thr)-Tn(Thr)-Tn(Thr)-G. Normalized data are plotted on a log 2 scale with a floor value of 7.2 (colored black).

Figure 3. Changes in overall immunoglobulin levels.

Overall levels of immunoglobulin (A  =  IgG, B  =  IgM, C  =  IgA) were measured in macaques (n = 30) at two time points – week 0 (prior to-vaccination and SIV challenge) and week 64 (after vaccination and SIV challenge). Dashed lines indicate the median values for each time point. P-values for comparison of the two time points were calculated with the Mann-Whitney test. Additionally, histograms show the fold changes in overall immunoglobulin (D  =  IgG, E  =  IgM, F  =  IgA) occurring after vaccination and SIV challenge.

Figure 4. Post-vaccination changes in anti-glycan antibodies.

Heat map showing changes in IgG (A) and IgM (B) circulating anti-glycan antibodies that occurred after vaccination (wk 38–wk 0). Rows correspond to individual macaques (n = 38) sorted by vaccine groups described in Table 1. Columns correspond to glycans grouped according to glycan family. Red indicates increases, and decreases are shown in blue. Non-significant changes (<4×) are white. As indicated by the arrows, Manα1-6Manβ had the most number of antibody changes, OSM had the largest increase in antibody levels, and LeY had the largest decrease. The dashed horizontal line marks the macaque with the large decline in anti-LeY IgM levels, and the solid line indicates the macaque with the largest OSM increase.

Figure 5. Early changes in anti-glycan antibody levels after SIV infection.

Heat map showing changes in IgG (A) and IgM (B) circulating anti-glycan antibodies that occurred after vaccinated macaques were challenged with SIV (wk 46–wk 38). Rows correspond to individual macaques (n = 38) sorted according to their ability to control SIV infection, as indicated by viral load measured at week 64 Columns are glycans grouped by category. Non-significant changes (<4×) have been colored white. Five macaques (animals 6, 12, 13, 19, and 43) with the most consistent Tn responses are marked with horizontal arrows. Vertical arrows indicate two Tn glycopeptides (solid arrow is Ac-S-Tn(Thr)-S-G-04 and dashed arrow is Ac-S-Tn(Thr)-V-G-04), which had changes occurring in the largest number of macaques (32%).

Figure 6. Later changes in anti-glycan antibody levels after SIV infection.

Post-infection changes in anti-glycan antibody levels were repeated for overall week 64 (22 weeks after challenge). Heat map showing changes in circulating IgG (A) and IgM (B) anti-glycan antibodies that occurred after vaccinated macaques were challenged with SIV (wk 64–wk 38). Rows correspond to individual macaques (n = 38) sorted according to their ability to control SIV infection, as indicated by viral load measured at week 64. Columns are glycans grouped by category. White indicates non-significant changes (<4×). Five macaques (animals 6, 12, 13, 19, and 43) with the most consistent Tn responses are marked with arrows.

Figure 7. Post-vaccination Tn responses.

Five of the 38 macaques showed increases in anti-Tn IgG (A) and/or IgM (B). For these 5 animals, blue bars indicate the average fold change for 23 low density Tn glycoproteins occurring within 8 weeks of SIV infection (overall week 46–week 38). Red bars show the fold change after 26 weeks of infection (overall week 64–week 38). The animals are ordered in increasing levels of viremia.