A novel, killed-virus nasal vaccinia virus vaccine

Abstract

Live-virus vaccines for smallpox are effective but have risks that are no longer acceptable for routine use in populations at minimal risk of infection. We have developed a mucosal, killed-vaccinia virus (VV) vaccine based on antimicrobial nanoemulsion (NE) of soybean oil and detergent. Incubation of VV with 10% NE for at least 60 min causes the complete disruption and inactivation of VV. Simple mixtures of NE and VV (Western Reserve serotype) (VV/NE) applied to the nares of mice resulted in both systemic and mucosal anti-VV immunity, virus-neutralizing antibodies, and Th1-biased cellular responses. Nasal vaccination with VV/NE vaccine produced protection against lethal infection equal to vaccination by scarification, with 100% survival after challenge with 77 times the 50% lethal dose of live VV. However, animals protected with VV/NE immunization did after virus challenge have clinical symptoms more extensive than animals vaccinated by scarification. VV/NE-based vaccines are highly immunogenic and induce protective mucosal and systemic immunity without the need for an inflammatory adjuvant or infection with live virus.

FIG. 1.

Complete virus inactivation with NE. (A) PRA of VV_WR. (B) Luciferase assay of VV_WR-Luc. Luciferase activity is presented in relative light units (RLU). In standardized assays, the limit of virus detection was <10 PFU for PRA and <4 PFU for the luciferase assay. (C) PCR analysis of lung DNA. Lanes: 1, DNA size marker; 2, primers, no DNA; 3, no Taq; 4, 10⁵/Fk lung DNA; 5 to 7, 10⁵/Fk/NE lung DNA; 8 to 10, 10⁵/NE lung DNA; 11, control (VV DNA mixed with lung DNA collected from tissue harvested 4 days after vaccination). Arrows indicate amplified viral template and primers. The limit of detection of this assay was <0.001 ng viral DNA. (D) In vivo bioluminescence imaging of mice after i.n. infection with live VV_WR-Luc and with 10⁵ PFU of NE-killed virus. Circles visible in some images indicate the regions of interest used for the photon flux analysis (Table 2).

FIG. 1.

Complete virus inactivation with NE. (A) PRA of VV_WR. (B) Luciferase assay of VV_WR-Luc. Luciferase activity is presented in relative light units (RLU). In standardized assays, the limit of virus detection was <10 PFU for PRA and <4 PFU for the luciferase assay. (C) PCR analysis of lung DNA. Lanes: 1, DNA size marker; 2, primers, no DNA; 3, no Taq; 4, 10⁵/Fk lung DNA; 5 to 7, 10⁵/Fk/NE lung DNA; 8 to 10, 10⁵/NE lung DNA; 11, control (VV DNA mixed with lung DNA collected from tissue harvested 4 days after vaccination). Arrows indicate amplified viral template and primers. The limit of detection of this assay was <0.001 ng viral DNA. (D) In vivo bioluminescence imaging of mice after i.n. infection with live VV_WR-Luc and with 10⁵ PFU of NE-killed virus. Circles visible in some images indicate the regions of interest used for the photon flux analysis (Table 2).

FIG. 2.

Immunogenicity of mucosal NE vaccine in mice. (A) Development of serum anti-VV IgG antibody response in mice vaccinated with various formulations of killed-virus vaccine: 10⁵/NE (•), 10³/NE (○), 10⁵/Fk/NE (▴), 10³/Fk/NE (▵), 10⁵/Fk (⧫), and 10³/Fk (⋄). Arrows indicate i.n. administrations of the vaccine. (Inset) Comparison of serum anti-VV IgG levels after one or three vaccinations with 10⁵/NE. Data are presented as mean individual anti-VV IgG concentrations ± SEM. (B) Secretory anti-VV IgA antibody in BAL fluid. Results are presented as mean concentrations (± SEM) of IgA obtained in assays performed with individual and pooled BAL fluids collected at 16 weeks.

FIG. 3.

Virus-neutralizing antibodies. Assays for virus neutralization were performed with both individual and pooled sera obtained after one, two, and three vaccinations. (Inset) Detection of virus-neutralizing activity in BAL fluids. Assays were performed with individual and pooled BAL fluids collected at the conclusion of the experiment at 16 weeks. Results were normalized and presented as NT₅₀s for the viral PRA.

FIG. 4.

VV-specific cellular immune responses. The IFN-γ expression in vitro in splenocytes stimulated with 10³ and 10⁴ PFU of live VV_WR. The data show a specific IFN-γ response to the virus in splenocytes from animals immunized with VV inactivated by NE.

FIG. 5.

VV-specific CD8 and CD4 responses. Splenocytes from mice immunized i.n. with NE-killed vaccine were analyzed for VV-specific responses 10 days after the second booster administration of vaccine. Frequencies of IFN-γ⁺ VV-specific CD8⁺ and CD4⁺ cells. Data are presented as mean values obtained from splenocytes from different groups of mice (n = 3) immunized with either nasal VV/NE or VV/scar ± SEM.

FIG. 6.

i.n. challenge with live VV. (A) Survival curves for mice vaccinated with 10⁵ PFU of killed VV_WR in various vaccine formulations, VV/NE, VV/Fk/NE, and VV/Fk, after i.n. challenge with 10 times the LD₅₀ of VV_WR-Luc. Significant protection was observed for the vaccinated animals compared to what was seen for control mice vaccinated with either NE alone or PBS. (B) Bioluminescence images of representative vaccinated (top) and control (bottom) mice challenged with a luciferase-producing VV. Images were recorded 2 to 5 days after challenge.

FIG. 7.

Comparison of IgG serum anti-VV titers with nasal vaccination using NE-killed virus and VV/scar. Mice were vaccinated with either three doses of VV/NE vaccine at 0, 3, and 8 weeks (VV/NE) or one VV/scar at week 0. VV/NE serum samples were analyzed at 3, 5, and 11 weeks, and VV/scar samples were analyzed at 3 weeks. Titers were assessed by ELISA at a 1:1,000 serum dilution to assure analysis in the linear range of the assay. (A) Titers in the first assay showed higher initial antibody concentrations in the mice immunized with scarification (P < 0.5). (B) Subsequent serial studies again showed a higher titer for scarification at 3 weeks but no significant difference at later time points (up to 11 weeks) after booster immunization with the VV/NE vaccine. Overall titers after week 5 were similar to those seen for naive animals who survived an LD₅₀ challenge with live virus (CTRL+, black bar).