Recombinant α-actinin subunit antigens of Trichomonas vaginalis as potential vaccine candidates in protecting against trichomoniasis

Abstract

Background: Human trichomoniasis caused by Trichomonas vaginalis is one of the most common sexually transmitted diseases with more than 200 million cases worldwide. It has caused a series of health problems to patients. For prevention and control of infectious diseases, vaccines are usually considered as one of the most cost-efficient tools. However, until now, work on the development of T. vaginalis vaccines is still mainly focused on the screening of potential immunogens. Alpha-actinin characterized by high immunogenicity in T. vaginalis was suggested as a promising candidate. Therefore, the purpose of this study was to evaluate the protective potency of recombinant α-actinin against T. vaginalis infection in a mouse intraperitoneal model.

Methods: Two selected coding regions of α-actinin (ACT-F, 14-469 aa and ACT-T, 462-844 aa) amplified from cDNA were cloned into pET-32a (+) expression vector and transfected into BL21 cells. After induction with IPTG and purification with electroelution, the two recombinant fusion proteins were emulsified in Freund's adjuvant (FA) and used to immunize BALB/C mice. Following intraperitoneal inoculation with T. vaginalis, the survival rate of mice was monitored for the assessment of protective potency. After immunization, the antibody level in mouse serum was assessed by ELISA, splenocyte proliferation response was detected with CCK8 and cytokines in the supernatant of splenocytes were quantified with a cytometric bead-based assay.

Results: We successfully obtained purified ACT-F (70.33 kDa) and ACT-T (61.7kDa). Both recombinant proteins could provide significant protection against T. vaginalis challenge, especially ACT-T (with 100% protection within one month). Meanwhile, high levels of specific total IgG and subtypes (IgG1 > IgG2a) were detected in sera from the immunized mice. Our results also revealed a statistically significant increase in splenocyte proliferation and related cytokine (IFN-γ, IL-6, IL-17A and IL-10) production after repeated stimulation with the corresponding antigens in vitro.

Conclusions: Immunization with both ACT-F and ACT-T could confer partial to complete protection and trigger strong Th1/Th2 mixed humoral and cellular immune responses in the mouse host. This suggested that recombinant α-actinin subunit antigens may be promising vaccine candidates against trichomoniasis.

Keywords: Alpha-actinin; Recombinant protein; Trichomonas vaginalis; Vaccine.

Fig. 1

Prokaryotic expression and purification of α-actinin. a SDS-PAGE analysis of prokaryotic expression of recombinant α-actinin peptides (ACT-F and ACT-T). IPTG-, non-induced; IPTG+, 0.6 mM IPTG induced for 4 h; purified, after electroelution purification. b Western blotting analysis using anti-His tag monoclonal antibody (at 1:1,000 dilution) and sera containing rabbit anti-sera (at 1:2,000 dilution). ACT-F, 70.33 kDa; ACT-T, 61.7 kDa

Fig. 2

Assessment of protective potency of α-actinin peptide vaccines in mice. BALB/C mice were immunized with ACT-F (high, n = 11; low, n = 12), ACT-T (high, n = 10; low, n = 15), whole-cell antigen (n = 10), PBS in adjuvant (FA-sham; n = 11), unimmunized (n = 17) and followed by intraperitoneal challenge with T. vaginalis (1 × 10⁷ per mouse) two weeks after last immunization. Mice were monitored daily to obtain the survival percentage

Fig. 3

Levels of IgG in the sera of mice immunized with specific antigens. a Total IgG titers exhibited, (b-d) total IgG (at 1:320,000 dilution), IgG subclasses (at 1:1,000 and 1:200,000 dilution) determined by OD450 nm value in the mice sera collected one day before each immunization as indicated. Each bar represents the mean value ± standard error (SEM) (n = 5). Significance was accepted by comparison with the unimmunized group (*P < 0.05; **P < 0.01; ***P < 0.001) or FA-sham group (^# P < 0.05; ^## P < 0.01; ^### P < 0.001) using statistical analyses

Fig. 4

Native α-actinin in T. vaginalis was recognized by serum from immunized mice. Immunofluorescence of native parasite α-actinin detected with mouse sera (at 1:2,000 dilution) from different groups: a T. vaginalis were incubated with pre-immunized mouse serum; b T. vaginalis were incubated with serum from FA-sham immunized mice; c T. vaginalis were incubated with serum from high dosage ACT-F immunized mice; d T. vaginalis were incubated with serum from high dosage ACT-T immunized mice serum. Goat-anti mouse IgG FITC (at 1:800 dilution) was used as secondary antibody. T. vaginalis α-actinin was labeled with FITC (in green) and DNA was stained with DAPI (in blue). Scale-bar: 10 μm

Fig. 5

Proliferation analysis of splenocytes from immunized mice in vitro. Splenocytes from ACT-F- high dosage, ACT-T- high dosage, FA-sham (PBS in adjuvant) and unimmunized groups were stimulated with ACT-F or ACT-T (10 μg/ml) for 68 h in vitro. Proliferation was determined using CCK8 cell counting kits. The stimulation index (SI) is calculated as the ratio of the proliferation of stimulated cells to non-stimulated cells in the same group. The data are the mean SI ± SEM from three individual mice from each group with three repeats (n = 3). Significance was accepted by comparison with the unimmunized (*P < 0.05; **P < 0.01; ***P < 0.001) and FA-sham groups (^# P < 0.05; ^## P < 0.01; ^### P < 0.001) using statistical analyses

Fig. 6

Cytokine secretion of splenocytes from immunized mice. Cytokines in supernatants were assayed in duplicate using CBA Kits (n = 3). The data are shown as mean value ± standard error (SEM) for antigen treatment. Significance was calculated by comparison with the unimmunized group (*P < 0.05; **P < 0.01; ***P < 0.001) or the FA-sham group (^# P < 0.05; ^## P < 0.01; ^### P < 0.001) using statistical analyses