High Titer Persistent Neutralizing Antibodies Induced by TSST-1 Variant Vaccine Against Toxic Shock Cytokine Storm

Abstract

Staphylococcal superantigen toxins lead to a devastating cytokine storm resulting in shock and multi-organ failure. We have previously assessed the safety and immunogenicity of a recombinant toxic shock syndrome toxin 1 variant vaccine (rTSST-1v) in clinical trials (NCT02971670 and NCT02340338). The current study assessed neutralizing antibody titers after repeated vaccination with escalating doses of rTSST-1v. At study entry, 23 out of 34 subjects (67.6%) had neutralizing antibody titers inhibiting T cell activation as determined by ³H-thymidine incorporation at a serum dilution of ≤1:100 with similar figures for inhibition of IL-2 activation (19 of 34 subjects, 55.9%) as assessed by quantitative PCR. After the first vaccination, numbers of subjects with neutralization titers inhibiting T cell activation (61.7% ≥ 1:1000) and inhibiting IL-2 gene induction (88.2% ≥ 1:1000) increased. The immune response was augmented after the second vaccination (inhibiting T cell activation: 78.8% ≥ 1:1000; inhibiting IL-2 induction: 93.9% ≥ 1:1000) corroborated with a third immunization months later in a small subgroup of subjects. Assessment of IFNγ, TNFα and IL-6 inhibition revealed similar results, whereas neutralization titers did not change in placebo participants. Antibody titer studies show that vaccination with rTSST-1v in subjects with no/low neutralizing antibodies can rapidly induce high titer neutralizing antibodies persisting over months.

Keywords: Staphylococcus; immunogenicity; neutralizing antibodies; toxic shock syndrome.

Figure 1

(A) Disposition of subjects. Trial flow chart of first-in-man and follow-up studies. Time tables indicate time periods of vaccination. At each time point sera were taken for binding and neutralizing antibody titer assessment. Vaccine doses are given for Study 1 and Study 3, number or participants per group are added in parentheses. (B) Persistence and booster-ability of antisera from invited participants of a follow-up study. Increase of binding titers in a log-lin scale of invited participants, who received a third immunization. Out of fifteen participants, who followed the invitation, fourteen were included in the per-protocol evaluation. One participant with a 100-fold increase 28 days after the booster decided to withdraw before the last time point. Additional sera were taken before (designated as after 2nd (II)), 28 days (after 3rd) and 6 months (after 3rd (II)) after the third immunization. p-values were calculated in a paired, two-tailed t-test (** p < 0.01, *** p < 0.001). Two grey arrows in the fifth column indicate the binding titers of the two non-responders finally displaying seroconversion.

Figure 2

Neutralization of T cell activation and cytokine induction by sera from study participants. Scattered dot plots with geometric means (including 95% CI) of neutralizing antibody titers of sera from immunized (A) and placebo (B) subjects throughout all time points (serum dilutions ≤100 in green, 300 in pink, 1000 in blue, 3000 in violet). Thirty-four subjects receiving the rTSST-1v and 12 placebo recipients (Al(OH)₃) were analyzed. One subject was a drop-out after the first vaccination. Twenty subjects were analyzed for persistence of neutralization titers. Fourteen subjects proceeded to the final part of the study, and one subject was a drop-out at the last study visit. Neutralizing sera from all vaccinated subjects (n = 33) are shown from before the first immunization (day 0/before 1st), after the first immunization (14 days / after 1st), twice after the second immunization (14 days (after 2nd) and 6 to 15 months (after 2nd II)). In the follow-up study, the schedule was set for 6 to 15 months after the second immunization (before 3rd), the mean elapse time being 294 days. Further values of titers were determined 28 days after the third immunization and six months after the third immunization (after 3rd II). p values were calculated by the paired, two-tailed Wilcoxon test (**** p < 0.0001, * p < 0.05). Geometric mean titers including 95% CI of neutralizing antibodies before the first and 14 days after the second immunization in immunized and placebo recipients determined by inhibition of T cell proliferation and cytokine gene induction are shown.

Figure 3

Neutralization of inflammatory cytokine induction by sera from study participants. Scattered dot plots with geometric means (including 95% CI) of neutralizing antibody titers of sera from immunized and placebo subjects (serum dilutions ≤100 in green, 300 in pink, 1000 in blue, 3000 in violet). Thirty-four subjects receiving the rTSST-1v and 12 placebo recipients (Al(OH)₃) were analyzed. One subject was a drop-out after the first vaccination. Neutralizing sera from all vaccinated subjects (n = 33) are shown from before the first immunization (day 0/before 1st), after the first immunization (14 days / after 1st) and twice after the second immunization (14 days/after 2nd). P values were calculated by the paired, two-tailed Wilcoxon test (**** p < 0.0001). Geometric mean titers including 95% CI of neutralizing antibodies before the first and 14 days after the second immunization in immunized and placebo recipients determined by inhibition of T cell proliferation and cytokine gene induction are shown.