Impact of ST-246® on ACAM2000™ smallpox vaccine reactogenicity, immunogenicity, and protective efficacy in immunodeficient mice

Abstract

Although a highly effective vaccine against smallpox, vaccinia virus (VV) is not without adverse events, some of which can be life-threatening, particularly in immunocompromised individuals. We have recently demonstrated that the immunogenicity and protective efficacy of Dryvax(®) in immunocompetent mice is preserved even when co-administered with ST-246, an orally bioavailable small-molecule inhibitor of orthopoxvirus egress and dissemination. In addition, ST-246 markedly reduced the reactogenicity of the smallpox vaccine ACAM2000 and the highly neurovirulent VV strain Western Reserve (VV-WR). Here, we evaluated the impact of ST-246 co-administration on ACAM2000 reactogenicity, immunogenicity, and protective efficacy in seven murine models of varying degrees of humoral and cellular immunodeficiency: BALB/c and B-cell deficient (JH-KO) mice depleted of CD4(+) or CD8(+) or both subsets of T cells. We observed that ST-246 reduced vaccine lesion severity and time to complete resolution in all of the immunodeficient models examined, except in those lacking both CD4(+) and CD8(+) T cells. Although VV-specific humoral responses were moderately reduced by ST-246 treatment, cellular responses were generally comparable or slightly enhanced at both 1 and 6 months post-vaccination. Most importantly, in those models in which vaccination given alone conferred protection against lethal VV challenge, similar levels of protection were observed at both time points when vaccination was given with ST-246. These data suggest that, with the exception of individuals with irreversible, combined CD4(+) and CD8(+) T-cell deficiency, ST-246 co-administered at the time of vaccination may help reduce vaccine reactogenicity--even in those lacking humoral immunity--without impeding the induction of protective immunity.

Figure 1. Effect of ST-246 on vaccine lesion formation and progression in immunodeficient mice

BALB/c (A) or B-cell deficient (JH-KO; B) mice were initially depleted of CD4⁺ and/or CD8⁺ T cells by i.p. injection of anti-CD4 and/or anti-CD8 mAbs for 3 consecutive days (days −5, −4, and −3) prior to vaccination (on day 0). Thereafter, cellular depletions were maintained for the duration of the experiment by once-weekly mAb injections (days +4, +11, etc). The mice were vaccinated by epidermal scarification of the tail with ~2.5×10⁵ PFU of ACAM2000 and treated by oral gavage with vehicle or 100 mg/kg of ST-246 for 14 consecutive days. Naïve (unvaccinated) and vaccinated non-depleted (N.D.) mice, which were not treated with antibody, served as controls. Photographs of the tails were taken on days 7, 14, 17, 21, and 28 post-vaccination to document vaccine-induced lesion severity. Although there were differences in the degree of lesion severity among mice within a group (n = 4–5), especially in CD8⁻CD4⁻ cohorts, representative tail lesions are shown.

Figure 2. Short- and long-term protective efficacy of ACAM2000 given in combination with ST-246 in immunodeficient mice

BALB/c (A-C;G-I) and JH-KO (D-F;J-L) mice were depleted of CD4⁺, CD8⁺, or both subsets of T cells, vaccinated, and concurrently treated with vehicle or ST-246 as described in Figure 1. One (A-F) or six months (G-L) post-vaccination, the mice were i.n. challenged with age-specific 10 LD₅₀ of VV-WR and monitored for survival (A,D,G,J), weight loss (B,E,H,K), and degree of clinical disease symptoms (C,F,I,L). For challenge at 1 month p.v., cellular depletion was maintained throughout the vaccination and challenge periods. For challenge at 6 months p.v., antibody treatment was discontinued on day 66 p.v. (although some CD8⁻CD4⁻ mice still had unhealed lesions), reinitiated five days prior to challenge, and continued throughout the monitoring period (i.e., 28 days). The graphs represent average values for 4-5 mice/group. Vaccinated mice treated with vehicle are indicated by open symbols while those treated with ST-246 are shown as closed symbols. ^‡ Naïve JH-KO mice were not included in the 6 months challenge study. ^§ In the CD8⁻CD4⁻/JH-KO group, there were only three vaccine/vehicle mice available for virus challenge since two mice in the group died from vaccine-induced systemic disease on days 41 and 45 p.v., while all five vaccine/ST-246 survived until and beyond cessation of depleting antibody treatment on day 66 p.v.

Figure 3. Inhibition of comet tail formation by EV-specific antibodies

Plasma samples were obtained from parallel groups of BALB/c (A-H) and JH-KO (I-J) immunodeficient mice described in Figure 2 that were sacrificed at one or six months post-vaccination. For the 1 month time point, cellular depletion was maintained until the day of sacrifice. For the 6 months time point, depleting antibody treatment was reinitiated five days prior to sacrifice. Pooled plasma from 4-5 mice/group were diluted 1:25 in BSC-40 cell cultures that were first infected with ~65 PFU of VV-IHD-J for 2 hours and overlayed with liquid medium. After 40 hours of incubation, the monolayers were stained with 0.1% crystal violet to visualize plaques and comet tails. Wells to which no plasma (M) or naïve BALB/c (K) or JH-KO (L) plasma were added, served as negative controls. Wells containing infected monolayers that were overlaid with 2% methylcellulose (N) were used as positive controls for complete inhibition of comet formation. ^‡ Indicate representative wells for all JH-KO groups including N.D./JH-KO, CD8⁻/JH-KO, CD4⁻/JH-KO, and CD8⁻CD4⁻/JH-KO since none of these groups inhibited comet formation and, thus, showed similar patterns. See Supplementary Table 2 for a qualitative scoring of the extent of comet tail inhibition.

Figure 4. Ex vivo analysis of the magnitude of VV peptide-specific CD8⁺ T-cell responses

Single-cell suspensions were prepared from spleens obtained from the same BALB/c (A) and JH-KO (B) immunodeficient mice described in Figure 3, which were sacrificed at one or six-months post-vaccination and concurrent treatment with vehicle or ST-246. Pooled splenocytes from 4-5 mice/group were consecutively stained with KYGRLFNEI (H-2K^d: A52R_75-83) peptide-loaded pentamer and anti-CD8 antibody and analyzed by flow cytometry. Dot plots represent CD8⁺ T cells subgated on live lymphocytes identified by FSC and SSC parameters. Naïve splenocytes were used as negative controls to set the pentamer⁺ gate. Percentages represent the frequency of pentamer⁺ cells in the CD8⁺ T-cell population. Although splenocytes from CD8⁻ and CD8⁻CD4⁻/BALB/c and JH-KO mice were also analyzed similarly, their plots are not shown, since the depletion of the CD8⁺ T-cell population was >99%.

Figure 5. Intracellular cytokine staining detection of VV-specific CD8⁺ and CD4⁺ T-cell responses

Splenocyte single-cell suspensions described in Figure 4 were stimulated in vitro with mock- or VV-WR-infected syngeneic A20 cells in the presence of IL-2 and Brefeldin A. After 16-18 hours of co-culture, the cells were surface stained with anti-CD4 and anti-CD8 antibodies, fixed, permeabilized, intracellularly stained with anti-IL-2 and -IFN-γ antibodies, and then analyzed by flow cytometry. Live lymphocytes were first identified and gated by FSC and SSC parameters and then subgated based on CD8 or CD4 expression. Panels depict the background (mock stimulated)-subtracted frequency of VV-specific CD8⁺ (A,B,E,F) or CD4⁺ (C,D,G,H) T cells expressing the indicated cytokines, as the arithmetic mean ± standard deviation of individually analyzed spleens (n = 4-5). These frequencies were <0.02% for both T-cell subsets in the spleens of naïve BALB/c and JH-KO mice (not shown). Although splenocytes from all of the immunodeficient groups were analyzed similarly, data corresponding to the depleted T-cell subset(s) is not shown due to >99% depletion efficiency. *P-value < 0.05.

Figure 6. Quantification of VV-specific secretion of IFN-γ (A,E), TNF-α (B,F), IL-2 (C,G), and IL-4 (D,H) by in vitro-stimulated splenocytes

Splenocyte single-cell suspensions described in Figure 4 were co-cultured with mock- or VV-WR-infected syngeneic A20 cells without the addition of BFA or IL-2. After 48 hours of stimulation, cell-free supernatants were collected and analyzed by T_H1/T_H2 cytokine CBA. The arithmetic mean ± standard deviation of background (mock stimulated)-subtracted values from individually analyzed spleens (n = 4–5) is shown. These values were less than the minimum quantifiable levels of the CBA assay (i.e., 20 pg/ml) in similarly stimulated splenocytes from naïve BALB/c and JH-KO mice (not shown). Although the VV-specific IL-5 secretion was also assayed, it was <20 pg/ml in all the indicated groups (not shown). *P-value < 0.05.