DNA-based vaccines protect against zoonotic schistosomiasis in water buffalo

Abstract

Schistosomiasis japonica is an endemic, zoonotic disease of major public health importance in China where water buffaloes account for approximately 75% of disease transmission. Interventions that reduce schistosome infection in water buffaloes will enhance their health simultaneously reducing disease transmission to humans. While chemotherapy has proved successful, it requires continued time consuming and expensive mass treatments. A more sustainable option would be development of vaccines that reduce transmission of S. japonicum from bovines to replace bovine chemotherapy. We performed two randomized double blind trials in water buffaloes to determine if DNA vaccines encoding triose-phosphate isomerase (SjCTPI), or the tetraspanin 23 kDa integral membrane protein (SjC23), alone or fused to bovine heat shock protein 70 (Hsp70) could induce a level of immunity conducive to long-term sustainable control. Groups of water buffaloes (15/group) received three intramuscular injections, 4 weeks apart. Booster immunizations were co-administered with a plasmid DNA encoding IL-12. Four weeks after the last injection, water buffaloes were challenged with 1000 cercariae, and vaccine efficacy analyzed 8 weeks later. Water buffaloes vaccinated with SjCTPI-Hsp70 or SjCTPI plasmids had worm burdens reduced by 51.2% and 41.5%, respectively. Importantly, fecal miracidial hatching was reduced by 52.1% and 33.2% respectively compared to control vaccinated water buffaloes. Vaccination with SjC23-Hsp70 and SjC23 plasmids reduced worm burdens by 50.9% and 45.5%, respectively, and fecal miracidial hatching by 52.0% and 47.4%. A mathematical model of schistosome transmission predicts that schistosome vaccines capable of reducing water buffaloes' fecal egg output by 45%, alone or in conjunction with praziquantel treatment, will lead to a significant reduction in transmission of schistosomiasis. Both DNA vaccines tested here exceed this hypothetical level. Indeed, mathematical modeling of SjCTPI-Hsp70 and SjC23-Hsp70 alone and in conjunction with human chemotherapy showed a significant reduction in transmission almost to the point of elimination.

Figure 1

Schematic representation of schistosome DNA vaccine constructs. DNA sequences encoding the antigens were cloned into the pVAX1 plasmid at the indicated restriction sites. In case of the Hsp70-fusion constructs, antigens were spaced from Hsp70 by two codons for the amino acids (Glycine-Serine) which were generated by the BamHI site used to join both sequences.

Figure 2

Vaccination protocol of water buffaloes. Water buffaloes were obtained from schistosome-free area, treated with ivermectin, quarantined for 4 weeks, tagged and randomly divided into different groups (15 animals/group). Water buffaloes were injected intramuscularly 3 times, four weeks apart, challenged with 1000 cercariae, and perfused 8 weeks later. Blood samples were collected from animals at the indicated time points. The different groups in trials 1 and 2 are indicated.

Figure 3

Analysis of the humoral immune response. Western Blot analysis of four individual serum samples obtained from vaccinated animals from each group prior to challenge infection (A and B). Animals immunized with SjC23 (A, lanes 1–4) and SjC23-Hsp70 (A, lanes 5–8), SjCTPI (B, lanes 1–4) and SjCTPI-Hsp70 (B, lanes 5–8), elicited specific antibody response. Sera from animals immunized with the control pVAX plasmid did not react with rSjC23 (A, lanes 9–12) or rSjCTPI (B, lanes 9–12). Levels (OD values) of specific anti-SjCTPI (C) and anti-SjC23 (D) total IgG antibodies for all individual animals were analyzed by ELISA. Serum samples were obtained at 2 days prior to prime (Wk. 4), two weeks after the prime (Wk. 6), two weeks post the first boost (Wk. 10), two weeks post the second boost (Wk. 14), and two days prior to perfusion (Wk. 24). Arrows indicate the time of the different immunizations as well as the challenge infection. Values are expressed as means ± SE of the OD values of all animals within the same group.

Figure 3

Analysis of the humoral immune response. Western Blot analysis of four individual serum samples obtained from vaccinated animals from each group prior to challenge infection (A and B). Animals immunized with SjC23 (A, lanes 1–4) and SjC23-Hsp70 (A, lanes 5–8), SjCTPI (B, lanes 1–4) and SjCTPI-Hsp70 (B, lanes 5–8), elicited specific antibody response. Sera from animals immunized with the control pVAX plasmid did not react with rSjC23 (A, lanes 9–12) or rSjCTPI (B, lanes 9–12). Levels (OD values) of specific anti-SjCTPI (C) and anti-SjC23 (D) total IgG antibodies for all individual animals were analyzed by ELISA. Serum samples were obtained at 2 days prior to prime (Wk. 4), two weeks after the prime (Wk. 6), two weeks post the first boost (Wk. 10), two weeks post the second boost (Wk. 14), and two days prior to perfusion (Wk. 24). Arrows indicate the time of the different immunizations as well as the challenge infection. Values are expressed as means ± SE of the OD values of all animals within the same group.

Figure 4

Mathematical modeling of the schistosome DNA vaccine constructs. The DNA vaccine constructs SjC23-Hsp70 (A) and SjTPI-Hsp70 (B) were modeled alone and as an intervention (in conjunction with human annual mass treatment and an initial water buffaloes treatment) and compared to a hypothetical vaccine providing 45% efficacy. The red lines represents the effect of a hypothetical vaccine providing 45% efficacy alone on human prevalence; the black lines represents the effect of SjC23-Hsp70 vaccine (A) and SjCTPI-Hsp70 vaccine (B) providing 48% and 52% efficacy respectively on human prevalence; the blue lines represents the effect of the of SjC23-Hsp70 vaccine (A) or SjCTPI-Hsp70 (B) in combination with an initial bovine mass treatment and annual mass treatment of humans for 10 years on human prevalence; and the green lines represents the effect of SjC23-Hsp70 vaccine (A) and SjCTPI-Hsp70 vaccine (B) in combination with an initial bovine mass treatment and annual mass treatment of humans for 10 years on water buffaloes prevalence.