Vaccine. 2026 Apr 08. pii: S0264-410X(26)00362-2. [Epub ahead of print]80
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BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan parasite capable of causing acute lethal disease and establishing lifelong chronic infection through tissue cyst formation. Development of an effective vaccine remains an important goal. Microneme protein 8 (MIC8), an invasion-associated antigen conserved across parasite lineages, represents a promising vaccine target.
METHODS: A heterologous DNA-protein prime-boost vaccination strategy targeting the MIC8 gene derived from the ME49 strain was evaluated in female BALB/c mice. Animals (n = 10 per group) were allocated to six groups: DNA + crude tachyzoite lysate antigen (TLA) boost, DNA alone, crude TLA alone, empty vector + TLA, empty vector alone, and phosphate-buffered saline (PBS). Immune responses were assessed by enzyme-linked immunosorbent assay (ELISA), IgG subclass profiling, antibody avidity assays, splenocyte proliferation, multi-colour flow cytometry with intracellular cytokine staining, and cytokine ELISA. Protective efficacy was evaluated by intraperitoneal challenge with 1 × 103 RH strain tachyzoites. Survival, clinical scores, and body weight were monitored for 130 days. Brain, liver, heart, and skeletal muscle tissues from survivors and controls were examined by haematoxylin and eosin staining, Dolichos biflorus agglutinin (DBA) lectin staining for cyst wall detection, immunohistochemistry, and quantitative PCR (qPCR) targeting the 529-bp repetitive element. In a separate challenge arm, protection against chronic cyst-forming infection was evaluated by oral challenge with 20 ME49 strain tissue cysts.
RESULTS: The DNA + TLA group elicited the highest MIC8-specific IgG levels (2.80 ± 0.01 mg/dL at week 6), a balanced IgG2a/IgG1 ratio (1.4 ± 0.2), elevated antibody avidity, and the greatest frequencies of activated CD4+CD44ʰʱᵍʰ (24.5 ± 2.1%) and CD8+CD44ʰʱᵍʰ (18.7 ± 1.8%) T cells with IFN-γ production. Following lethal RH challenge, the DNA + TLA group achieved 100% survival over 130 days, compared with 80% for DNA alone, 60% for crude TLA alone, and 0% for all control groups (median survival 10-12 days; log-rank p < 0.001). Pairwise comparisons showed DNA + TLA was statistically superior to crude TLA alone (p = 0.038) but not to DNA alone (p = 0.317). Following oral ME49 cyst challenge, vaccinated animals remained clinically stable throughout the 130-day observation period. At study endpoint, brain, liver, heart, and skeletal muscle tissues from vaccinated survivors showed no detectable cyst wall structures by DBA lectin staining, minimal inflammatory infiltrates, and negative qPCR signals, whereas all control tissues were strongly positive.
CONCLUSIONS: A MIC8-based heterologous DNA prime-crude TLA boost vaccination strategy induced strong antigen-specific humoral and cellular immune responses and conferred significant protection against lethal T. gondii RH strain challenge in BALB/c mice. The absence of detectable cysts and parasite DNA in vaccinated survivors at the examined endpoint suggests marked suppression of chronic parasite establishment under the conditions tested. Whilst the use of crude TLA for boosting precludes definitive attribution of protection solely to MIC8, the superior efficacy of the combined regimen over either component alone supports a synergistic contribution of MIC8-directed priming. These findings support further investigation of MIC8 and heterologous prime-boost strategies for toxoplasmosis vaccine development.
Keywords: DNA vaccine; MIC8; Prime–boost; Protective immunity; Tissue cysts; Toxoplasma gondii