One time intranasal vaccination with a modified vaccinia Tiantan strain MVTT(ZCI) protects animals against pathogenic viral challenge

Abstract

To combat variola virus in bioterrorist attacks, it is desirable to develop a noninvasive vaccine. Based on the vaccinia Tiantan (VTT) strain, which was historically used to eradicate the smallpox in China, we generated a modified VTT (MVTT(ZCI)) by removing the hemagglutinin gene and an 11,944bp genomic region from HindIII fragment C2L to F3L. MVTT(ZCI) was characterized for its host cell range in vitro and preclinical safety and efficacy profiles in mice. Despite replication-competency in some cell lines, unlike VTT, MVTT(ZCI) did not cause death after intracranial injection or body weight loss after intranasal inoculation. MVTT(ZCI) did not replicate in mouse brain and was safe in immunodeficient mice. MVTT(ZCI) induced neutralizing antibodies via the intranasal route of immunization. One time intranasal immunization protected animals from the challenge of the pathogenic vaccinia WR strain. This study established proof-of-concept that the attenuated replicating MVTT(ZCI) may serve as a safe noninvasive smallpox vaccine candidate.

Fig. 1

Schematic representation of the MVTT_ZCI genome. The 11,944 bp genomic deletion was found in the left terminal region of the viral genome. This deletion was confirmed by sequence analysis. The letters indicate the HindIII fragments. The vaccinia gene names are based on the sequence of vaccinia Copenhagen strain. The inset shows the results of Western blot analysis, which confirms the loss of the HA gene.

Fig. 2

Analysis of VTT quasispecies. To understand the source of the 11,944 bp genomic deletion, 20 randomly selected clones were purified from VTT. A PCR-scanning method was used to analyze the VTT quasispecies using 7 pairs of primers. Each pair of primers specific target one VTT gene open reading frame in the left terminal flank region from C7L to K8R. If a deletion occurs, the PCR product specific for the gene will be absent. We found that nine out of the 20 clones contained various gene deletions including 1, 2, 6, 13, 14, 15, 16, 17 and 20.

Fig. 3

Host cell range of MVTT_ZCIin vitro. (A) The replication kinetics of MVTT_ZCI was determined in the five cell lines indicated. Confluent cells were infected at a MOI of 0.05 with MVTT_ZCI or VTT viruses. The viral replication titer was measured on permissive Vero cells after absorption (0), 24, 48, and 72 h p.i. The experiments were repeated twice with similar results obtained. (B) Cell-to-cell spread of MVTT_ZCI was determined in the five cell lines tested. The indicated cells were infected with 100 PFU of MVTT_ZCI or VTT, fixed at 48 h p.i. and then immunostained with anti-VTT specific polyclonal antibody. The panels show representative fields at an approximately 100× magnification.

Fig. 4

Virulence of MVTT_ZCI in mice after intranasal and intracranial inoculations. (A) Groups of five BALB/c mice (5-week-old) were inoculated intranasally with 10⁶, 10⁵ and 10⁴ PFU of MVTT_ZCI or VTT in 30 μl of PBS on day 0 (arrow), respectively. Mice inoculated with PBS served negative controls. The body weight changes were represented by the mean values of each group of mice p.i. overtime. The error bar indicates the standard deviation (SD) of animals from each group. (B) Six mice per dilution group (3-week-old) were inoculated intracranially with 5-fold diluted MVTT_ZCI or VTT, respectively. The percentage of animals surviving was determined over 30 days observation p.i. None of mice died in MVTT_ZCI inoculated groups. (C) Groups of SCID mice (4 mice each group) were infected i.p. with MVTT_ZCI (10⁶ and 10⁷ PFU) or VTT (10³, 10⁴, 10⁵ and 10⁶ PFU). Mice inoculated with PBS were included as controls. The body weights of mice were measured individually and the mean values of each group were plotted. “†” indicates the loss of mice from infectious mortality or sacrifice due to 25% of body weight loss.

Fig. 5

Replication kinetics of MVTT_ZCI in mouse brain. Groups of 10 BALB/c mice (3-week-old) were inoculated intracranially with the indicated doses of VTT (A) or MVTT_ZCI (B), respectively. Another group of 10 SCID mice were given MVTT_ZCI (C). Two mice in each group were sacrificed daily during the first 5 days p.i. The titer of virus in brain homogenates was determined in Vero cells using a plaque-forming assay. The error bar indicates the standard deviation (SD) of animals from each group.

Fig. 6

Protection of mice against pathogenic vaccinia WR strain challenge. Groups of five BALB/c mice (5-week-old) were immunized once with 10⁶ PFU (A) or 10⁴ PFU (B) of MVTT_ZCI or VTT strain via indicated routes, respectively. Mice received PBS were included as controls. Thirty days post-immunization, mice were challenged intranasally with a lethal dose (10⁶ PFU, equivalent to over 100 LD₅₀) of WR strain. Mice that lose 25% of body weight were sacrificed according to the standard operating procedure. The body weight changes were represented by the mean values of each group of mice p.i. overtime. The error bar indicates the standard deviation (SD) of animals from each group.