A single dose recombinant AAV based CHIKV vaccine elicits robust and durable protective antibody responses in mice

Abstract

Background: Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that is responsible for Chikungunya fever, which is characterized by fever, rash, and debilitating polyarthralgia. Since its re-emergence in 2004, CHIKV has continued to spread to new regions and become a severe health threat to global public. Development of safe and single dose vaccines that provide durable protection is desirable to control the spread of virus. The recombinant adeno-associated virus (rAAV) vectors represent promising vaccine platform to provide prolonged protection with a single-dose immunization. In this study, we developed a rAAV capsid serotype 1 vector based CHIKV vaccine and evaluated its protection effect against CHIKV challenge.

Methodology: The recombinant AAV1 encoding the full-length structural proteins of CHIKV (named as rAAV1-CHIKV-SP) was generated in vitro by transfecting the plasmids of AAV helper-free system into HEK-293T cells. The safety and immunogenicity of rAAV1-CHIKV-SP were tested in 4-week-old C57BL/6 mice. The antibody responses of the mice receiving prime-boost or single-dose immunization of the vaccine were determined by ELISA and plaque reduction neutralizing test. The immunized mice were challenged with CHIKV to evaluate the protection effect of the vaccine.

Conclusions: The rAAV1-CHIKV-SP showed remarkable safety and immunogenicity in C57BL/6 mice. A single dose intramuscular injection of rAAV1-CHIKV-SP elicited high level and long-lasting antibody responses, and conferred complete protection against a heterologous CHIKV strain challenge. These results suggest rAAV1-CHIKV-SP represents a promising vaccine candidate against different CHIKV clades with a simplified immunization strategy.

Fig 1. Construction and characterization of rAAV-CHIKV-SP.

(A) The schematic representation of the recombinant genome and the processes of production and transduction of rAAV-CHIKV-SP. (B and C) The expression of CHIKV-E2 protein in pAAV-CHIKV-SP transfected HEK-293T cells at 48 hpt analyzed by immunostaining IFA (B) and western blot assay (C), respectively. (D) The genome titers of rAAV-CHIKV-SP produced by different AAV serotypes determined by qPCR. (E and F) The expression of CHIKV-E2 protein in HEK-293T cells infected with rAAV-CHIKV-SP at an MOI of 10⁵ detected by IFA (E) and western blot (F), respectively, at 72 hpi.

Fig 2. Antibody responses of C57BL/6 mice receiving prime-boost immunization of rAAV-CHIKV-SP.

(A) Experimental schedule. Four-to-six weeks old C57BL/6 female mice were IM injected with 5 × 10¹⁰ GC of rAAV1-CHIKV-SP, followed by boosting with the same dose of rAAV9-CHIKV-SP on day 21. Mice given 5 × 10¹⁰ GC rAAV1-eGFP were used as empty vector control and mice given PBS were set as mock. The serum samples from each mouse harvested on day 14 and 35 were subjected to ELISA and PRNT assays. The mice were challenged with 2.5×10⁵ PFU CHIKV ECSA strain virus through s.c. injection in feet on day 38, viremia was determined, and the symptoms post-challenge were recorded at the indicated times post challenge. (B) Body weight change of the immunized mice compared with the PBS control group within 2 weeks after the primary immunization. (C and D) The titers of CHIKV-specific antibodies (C) and neutralizing antibodies (D) from the serum samples determined by ELISA and PRNT, respectively. (E to G) IgG subtype analysis by ELISA. The serum samples from each group were subjected to ELISA assay to measure the levels of IgG2c (E) and IgG1 (F), and IgG2c/IgG1 ratios (G) were calculated and presented. The dashed lines in panels B, C, D, and E represent the limits of detection. Data were represented as the mean ± standard deviation (n = 5 in each group). * p < 0.05, **p < 0.01, **** p < 0.0001. n.s., no statistical difference.

Fig 3. Prime-boost immunization of rAAV-CHIKV-SP protects C57BL/6 mice from CHIKV challenge.

(A) Viremia post-challenge. Viremia of each mouse from day 1 to day 3 post-challenge was measured by plaque assay. (B) Footpad swelling examination after 14 days of challenge. (C) Representative images of the injected feet of the mice from each group on day 5 post-challenge. (D and E) The titers of CHIKV-specific antibodies (D) and neutralizing antibodies titers (E) on day 0 and day 28 post-challenge. The dashed lines in panels A, D, E represent the limits of detection. Data represent the mean ± standard deviation (n = 5 in each group). * p < 0.05, **p < 0.01, **** p < 0.0001.

Fig 4. Antibody responses of a single-dose immunization with rAAV1-CHIKV-SP in C57BL/6 mice.

(A) Experimental schedule. C57BL/6 mice (4–6 weeks old) were IM injected with different dosages (5 × 10⁸ GC, 5 × 10⁹ GC, or 5 × 10¹⁰ GC) of rAAV1-CHIKV-SP once. The serum samples on day 14 and 28 post-immunization were subjected to ELISA and PRNT assays. The mice were challenged with 2.5×10⁵ PFU CHIKV ECSA strain virus through s.c. injection in feet on day 30 post-immunization, viremia was determined, and the symptoms post-challenge were recorded at the indicated times post challenge. (B and C) The titers of CHIKV-specific antibodies (B) and neutralizing antibodies (C) from the serum samples determined by ELISA and PRNT, respectively. (D to F) IgG subtype analysis by ELISA. The levels of IgG2c (D) and IgG1 (E) were determined by ELISA, and IgG2c/IgG1 ratios (F) were calculated. Data represent the mean ± standard deviation (n = 5 in each group). * p < 0.05, **p < 0.01, **** p < 0.0001. n.s., no statistical difference.

Fig 5. A single-dose immunization with rAAV1-CHIKV-SP protects C57BL/6 mice from CHIKV infection.

(A) The viremia of mice receiving single-dose injection of 5 × 10⁸ GC, 5 × 10⁹ GC, or 5 × 10¹⁰ GC of rAAV1-CHIKV-SP respectively in 3 days post-challenge. (B) The footpad swelling measurements for 15 days after challenge. (C) Representative images of the injected feet of the mice from each group on day 5 post-challenge. The dashed lines in panels A represent the limit of detection. Data represent the mean ± standard deviation of 5 mice at each time point in each group. The asterisks denote statistical differences between the indicated groups. **** p < 0.0001; n.s., no statistical difference.

Fig 6. Long-term antibody responses induced by a single-dose of rAAV1-CHIKV-SP.

Groups of 4–6 weeks old C57BL/6 mice were given a single-dose IM injection of 5 × 10¹⁰ GC rAAV1-CHIKV-SP or rAAV1-eGFP or PBS. The serum samples of mice from each group were collected at 2, 4, 6, 8, 10 and 12 weeks after immunization, and subjected to ELISA and PRNT assays to quantify the levels of CHIKV-specific antibodies (A) and neutralizing antibodies (B), respectively. The dashed lines represent the limit of detection. Data represent the mean ± standard deviation of 5 mice at each time point in each group. The asterisks denote statistical differences between the indicated groups. * p < 0.05, **p < 0.01, **** p < 0.0001.