Development of a new hydrogen peroxide–based vaccine platform

Abstract

Safe and effective vaccines are crucial for maintaining public health and reducing the global burden of infectious disease. Here we introduce a new vaccine platform that uses hydrogen peroxide (H(2)O(2)) to inactivate viruses for vaccine production. H(2)O(2) rapidly inactivates both RNA and DNA viruses with minimal damage to antigenic structure or immunogenicity and is a highly effective method when compared with conventional vaccine inactivation approaches such as formaldehyde or β-propiolactone. Mice immunized with H(2)O(2)-inactivated lymphocytic choriomeningitis virus (LCMV) generated cytolytic, multifunctional virus-specific CD8(+) T cells that conferred protection against chronic LCMV infection. Likewise, mice vaccinated with H(2)O(2)-inactivated vaccinia virus or H(2)O(2)-inactivated West Nile virus showed high virus-specific neutralizing antibody titers and were fully protected against lethal challenge. Together, these studies demonstrate that H(2)O(2)-based vaccines are highly immunogenic, provide protection against a range of viral pathogens in mice and represent a promising new approach to future vaccine development.

Figure 1

H₂O₂ inactivates viruses without substantial damage to antigenic epitopes. (a) H₂O₂ was used to inactivate a group of viruses including YFV, WNV, LCMV, VV and monkeypox virus (MPV). After 2 h of incubation with or without H₂O₂, infectious virus was measured by plaque assay. The limit of detection is indicated by the dashed line. (b,c) Purified YFV was treated with H₂O₂, β-propiolactone, or formaldehyde (Form.) for 2 h (b) or 16 h (c) at 20–24 °C and compared to untreated, live virus for retained antigenicity. ELISA plates were coated with each purified virus preparation, and screened with convalescent serum from YFV-infected mice (n = 3). ELISA results were normalized to the levels observed when live virus was used as antigen (that is, percentage of live virus signal), and error bars represent s.d.

Figure 2

Vaccination with H₂O₂-LCMV induces multifunctional, cytolytic CD8⁺ T cells that protect against chronic virus infection. (a) BALB/c mice were infected with LCMV-Armstrong or vaccinated with H₂O₂-LCMV (n = 6 mice per group from three experiments). At 8 d after infection or vaccination, CD8⁺ T cells were stimulated with NP118 peptide and analyzed by flow cytometry for IFN-γ, TNF-α, and IL-2 production. Numbers in the top quadrants of the dot plots show the percentage of T cells producing the indicated cytokines after background subtraction, and the numbers in parentheses represent the percentage of T cells that are IFN-γ⁺TNF-α⁺ or IFN-γ⁺IL-2⁺. (b) The percentage of LCMV-specific IFN-γ⁺CD8⁺ T cell responses (top right quadrant), as measured at 28 d after primary infection or H₂O₂-LCMV vaccination (top) or 4 d after challenge (bottom) with LCMV-Armstrong (n = 3 or 4 mice per group). (c) Granzyme B expression assessed directly ex vivo in NP118-tetramer⁺CD8⁺ T cells from representative LCMV-Armstrong infected mice (live) or H₂O₂-LCMV–vaccinated mice (H₂O₂) at 28 d after infection or vaccination or at 4 d after challenge with LCMV-Armstrong (day 32). (d) In vivo lysis of peptide-coated targets at 8 d after H₂O₂-LCMV vaccination or LCMV-Armstrong infection, compared to naïve controls (n = 4 mice per group). Error bars represent s.d. (e) At 28 d after LCMV-Armstrong infection or at 7 d or 28 d after H₂O₂-LCMV vaccination, mice were challenged with LCMV clone 13, and viremia was monitored by plaque assay (n = 4 mice per group).

Figure 3

Induction of protective orthopoxvirus-specific neutralizing antibody responses following H₂O₂-VV immunization. (a) BALB/c mice (n = 4 per group) were vaccinated with live VV, MVA or purified VV inactivated with ultraviolet light (UV), 1% formaldehyde (Form.) for 2 h or 1% H₂O₂ for 2 h. Each group of mice was boosted 28 d later with the same vaccine preparation, and neutralizing antibody titers (NT₅₀) were determined 28 d after secondary immunization. The black bars indicate group geometric mean NT₅₀ titers with statistical comparisons determined using analysis of variance (ANOVA). (b) Naïve mice or mice that received primary H₂O₂-VV immunization 28 d previously (n = 6–8 mice per group) were challenged intranasally with 10 LD₅₀ of VV.

Figure 4

Vaccination with H₂O₂-WNV induces strong neutralizing antibody responses and protects against lethal WNV infection. (a) BALB/c mice (n = 4 mice per group) were vaccinated with H₂O₂-WNV or the Ft. Dodge WNV Innovator horse vaccine and boosted with the same vaccine at 28 d after primary vaccination. WNV-specific IgG responses ± the s.e.m. were determined by ELISA. (b) Virus-specific neutralizing antibody levels were determined for mice vaccinated with H₂O₂-WNV (2 months after booster vaccination) and compared to the levels observed after natural WNV infection in human subjects (~1 year after exposure). (c) Naïve mice and mice vaccinated with H₂O₂-WNV or Ft. Dodge WNV Innovator (75 d after booster vaccination) were challenged with 20 LD₅₀ of WNV-NY99 (n = 4 mice per group). (d) Naïve mice received immune serum from H₂O₂-WNV-vaccinated mice or naïve serum from unvaccinated mice 1 d before challenge with 20 LD₅₀ of WNV-NY99 (n = 3–5 mice per group).