A protein-based smallpox vaccine protects non-human primates from a lethal monkeypox virus challenge

Abstract

Concerns about infections caused by orthopoxviruses, such as variola and monkeypox viruses, drive ongoing efforts to develop novel smallpox vaccines that are both effective and safe to use in diverse populations. A subunit smallpox vaccine comprising vaccinia virus membrane proteins A33, B5, L1, A27 and aluminum hydroxide (alum) ± CpG was administered to non-human primates, which were subsequently challenged with a lethal intravenous dose of monkeypox virus. Alum adjuvanted vaccines provided only partial protection but the addition of CpG provided full protection that was associated with a more homogeneous antibody response and stronger IgG1 responses. These results indicate that it is feasible to develop a highly effective subunit vaccine against orthopoxvirus infections as a safer alternative to live vaccinia virus vaccination.

Fig. 1

Kaplan-Meier survival analyses and pock lesion summary. (A) Kaplan-Meier survival plots following MPXV challenge for the 3-dose study with 5 monkeys/group. Solid line with squares: Dryvax (Group 2), ABLA/CpG/alum (Groups 3 and 4), and ABL/CpG/alum (Group 6); dashed line with triangles: ABLA/alum (Group 5); dotted line with circles: CpG/alum control (Group 1). (B) Frequency of each lesion category for vaccination groups at days 0–27 post-challenge. The upper and lower quartiles of lesion frequencies for numeric lesion data were calculated and plotted. The 25th (1 to ≤7 lesions) and 75th percentiles (≥241 lesions) were used to recode the numeric lesion data into low (≤25th), medium (25–75th) and high (≥75th) lesion categories. For this analysis, lesion counts that were recorded as “TNTC” (too numerous to count) were set at ≥500 lesions. High lesions (≥241) were observed for all animals in Group 1 by day 9 post-challenge. High lesions were observed in 1 of 5 animals in Group 5 by days 9 and 2 of 5 animals in Group 6 by day 12. All animals in Group 3 experienced low (≤7) to medium (<241) lesions by day 9. All lesions of surviving animals in Groups 2–6 were healed by day 24, post-challenge. C. Kaplan-Meier survival plots following MPXV challenge for the 2-dose study with 3 monkeys/group. Solid line with squares: Dryvax (Group B) ABLA/CpG/alum (Groups C and D); dotted line with circles: CpG/alum control (Group A).

Fig. 2

Viral load on days 0–27 post-challenge. Graphed are the post-challenge viral load by group and monkey. The detection limit of the viral load assay was 5000 genome copies/mL blood. Median VL and inter-quartile ranges were determined and Dunn's multiple comparison test was performed on vaccination pairs where a significant main effect of vaccination groups was found. A higher viral load was found for Group 1 compared to (i) Group 2 (Dryvax) at days 3–15 (P < 0.05); (ii) Group 3 (ABLA(100)/CpG/alum) at days 3, 6, and 15 (P < 0.05); and (iii) Group 4 (ABLA(20)/CpG/alum) at days 3 and 15 (P < 0.05). *P < 0.05, ^P < 0.01, ⁺P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 3

Relationship between PRNT₅₀, lesion number, and viral load. Post-challenge lesion counts (open circles) and viral loads (solid triangles) at day 6 (A) and day 9 (B) are plotted against the log of the pre-challenge PRNT₅₀. Various colored symbols represent each different vaccination group: Group 1 (adjuvant only) black; Group 2 (Dryvax) blue; Group 3 (ABLA(100)/CpG/alum) red; Group 4 (ABLA(20)/CpG/alum) purple; Group 5 (ABLA/alum) green; Group 6 (ABL/CpG/alum) orange. For this analysis, “TNTC” lesions were set at 500 lesions. Correlations between pre-challenge neutralization (log[PRNT₅₀]) and day 6 lesions (r = −0.5433 P = 0.0019) and viral loads (r = −0.3997 P = 0.0287) and day 9 lesions (r = −0.3929 P = 0.0318) and viral loads (r = −0.3928 P = 0.0318) were statistically significant.

Fig. 4

Protein vaccines adjuvanted with CpG/alum provide higher IgG1 to IgG2 ratios that result in more consistent complement-enhanced neutralization of EV. (A) IgG1 and IgG2 isotype responses to B5 in Group 3 (ABLA(100)/CpG/alum) at day 98 (2 weeks after the second boost). (B) IgG1 and IgG2 isotype responses to B5 in Groups 5 (ABLA/alum) at day 98 (2 weeks after the second boost). Solid lines with solid symbols: IgG1 responses. Dashed lines with open symbols: IgG2 responses. (C) EV neutralization by NHP sera on day 98 (2 weeks after the second boost) in the absence and presence of complement (C′). Error bars represent standard deviation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)