Smallpox vaccine safety is dependent on T cells and not B cells

Abstract

The licensed smallpox vaccine, ACAM2000, is a cell culture derivative of Dryvax. Both ACAM2000 and Dryvax are administered by skin scarification and can cause progressive vaccinia, with skin lesions that disseminate to distal sites. We have investigated the immunologic basis of the containment of vaccinia in the skin with the goal to identify safer vaccines for smallpox. Macaques were depleted systemically of T or B cells and vaccinated with either Dryvax or an attenuated vaccinia vaccine, LC16m8. B cell depletion did not affect the size of skin lesions induced by either vaccine. However, while depletion of both CD4(+) and CD8(+) T cells had no adverse effects on LC16m8-vaccinated animals, it caused progressive vaccinia in macaques immunized with Dryvax. As both Dryvax and LC16m8 vaccines protect healthy macaques from a lethal monkeypox intravenous challenge, our data identify LC16m8 as a safer and effective alternative to ACAM2000 and Dryvax vaccines for immunocompromised individuals.

Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2011.

Figures

Figure 1.

B cell depletion in rhesus macaques. A, Study design: 6 macaques were vaccinated with LC16m8 (n = 3) group1 or Dryvax (n = 3) group 2, in the absence of antibody treatment. B, Six macaques were given α-CD20 antibodies 6 days prior and again 6 days post-vaccination with LC16m8 (n = 3) group 3 or Dryvax (n = 3) group 4. C, Flow cytometric dot plots showing the frequency of CD20 expressing cells at baseline (day−27), day −1, and day 27, in control and antibody treated animals. D, The absolute number of CD20-expressing cells before and after vaccination in antibody-treated and untreated animals. E–F, Flow cytometric dot plots showing the frequency of CD20+ cells in the bronchial alveolar lavage (BAL) and lymph nodes (LN) (top) and the average frequency of CD20+ cells before and after vaccination in controls (E) and α-CD20 treated macaques (***) represents P < .001 (F). G, Dryvax-vaccinated (right) animals had significantly higher neutralizing antibody titers to vaccinia (NYCBH strain) compared with LC16m8-vaccinated (left), assessed at 2 weeks post-vaccination or at time of death (sac) (P = .018 and P = .001, respectively). α-CD20 treatment caused a significant reduction in anti-vaccinia neutralizing antibody titer (P = .0027 and P < .025).

Figure 2.

LC16m8 and Dryvax induced skin lesions in B cell–depleted animals. A, Photographs of the primary skin lesion after vaccination in two untreated control macaques vaccinated with LC16m8 or Dryvax. B, Mean skin lesion size and time to resolution in untreated LC16m8 and Dryvax vaccinated macaques. C, Photgraphs of the skin lesions after vaccination with LC16m8 or Dryvax in B cell–depleted animals. D, Mean skin lesion size and time to resolution in B cell–depleted macaques vaccinated with LC16m8 and Dryvax. E, G, Photographs of the vaccine induce lesion (top) and the mean size of the skin lesion (bottom) in a Dryvax vaccinated animal P104 (E) and an LC16m8 vaccinated animal P102 (G). F, H, Absolute number of CD4+, CD8+, and CD20+ cells/mm3 in a Dryvax vaccinated animal P104 (F) and a LC16m8 vaccinated animal P102 (H).

Figure 3.

α-CD4 and α-CD8 treatment induces T cell depletion in the blood and tissues of rhesus macaques. A, Study design and antibody administration schedule (top), representative flow cytometric dot plots showing the frequency of CD4+ and CD8+ T cells in blood of control untreated animals and T cell–depleted macaques before and after vaccination. B–D, Mean absolute numbers of CD4+ T cells/mm3 (B) CD8+ T cells/mm3 (C), and CD3+ cells/mm3 (D), in the blood of antibody treated and control macaques before and after vaccination. E, Representative flow cytometric plots showing the frequency of CD3+ cells in the LN and BAL before (day −27) and after (day −1) α-CD4 and -CD8 treatment. F, Mean percentage of CD3+ cells in the LN and BAL. α-CD4 and α-CD8 treatment caused a significant depletion of CD3+ cells from the LN and BAL P = .018 and P = .022.

Figure 4.

T cell depletion favors increased lesion size and disseminating lesions in Dryvax vaccinated animals. A, Photographs of LC16m8 and Dryvax induced primary lesions in α-CD4 and α-CD8 treated macaques. B, Mean lesion size and resolution time following α-CD4 and α-CD8 treatment and vaccination with LC16m8 and Dryvax. C, Satellite lesions (arrows) surrounding the primary Dryvax vaccination site in one CD4 and CD8 depleted macaque P100. D, Lesions disseminating from the primary vaccination site, between the scapulas, to a distal site under the arm in one Dryvax vaccinated CD4 and CD8 depleted macaque M884. E, Anti-vaccinia neutralizing antibody titers in LC16m8-vaccinated (left), and Dryvax-vaccinated (right) animals, assessed at 2 weeks post-vaccination or at time of death (sac). Macaques were either untreated (control) or treated with α-CD4 and α-CD8 depleting antibodies.

Figure 5.

T cell depletion is a correlate of the size of Dryvax-induced but not LC16m8-induced skin lesions. A–C, Spearman ranked correlations between the number of CD20+ cells (A), CD4+ (B), and CD8+ (C) cells/mm3 and the size of either LC16m8 or Dryvax-induced skin lesions.

Figure 6.

LC16m8 and Dryvax protect cynomologus macaques from a lethal monkeypox intravenous challenge. A, Study design: 60 days post-scarification with LC16m8, Dryvax or PBS, macaques were challenged with monkeypox and monitored for 45 days. B, Monkeypox specific T cell responses after vaccination and monkeypox challenge in LC16m8 vaccinated (n = 14, left) Dryvax vaccinated (n = 4, middle) and unvaccinated macaques (n = 6, right) measured by IFN-γ-specific ELISPOTs. C, Monkeypox neutralizing antibody titers measured in vaccinated animals 2½ weeks prior to monkeypox challenge, ie, 42 days post-vaccination. D, Monkeypox DNA measured by quantitative PCR in the blood of LC16m8 or Dryvax vaccinated and unvaccinated macaques. E, Percentage of animals surviving after monkeypox challenge. A Fisher exact test demonstrated that the difference in survival between vaccinated and unvaccinated animals was statistically significant P < .0001(E).