Optimized GAPDH-truncated immunogen of Streptococcus equi elicits an enhanced immune response and provides effective protection in a mouse model.

Strangles is an acute and frequently diagnosed infectious disease caused by Streptococcus equi subsp. equi. Infection with this pathogen can cause grave losses to the equine industry. The present work investigates glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important surface-localized virulence factor of S. equi, to determine whether it could be developed into an efficacious and suitable subunit vaccine against strangles. Two different recombinant fragments of S. equi GAPDH, namely, GAPDH-L and GAPDH-S, were constructed and expressed. Further, the antigenicity and immunogenicity of these two recombinant proteins were compared and evaluated in a mouse model. Our results revealed that immune responses were efficiently induced by the proteins in immunized mice. Remarkably, higher survival rates and significantly lower bacterial loads in the lung, liver, kidney, and spleen were observed in the GAPDH-S group compared with the GAPDH-L group after challenge with S. equi. High levels of specific antibodies, elevated antibody titers, and increased proportions of CD8 + T cells further indicated that GAPDH-S elicited better humoral and cellular immune responses than GAPDH-L. Furthermore, the induction of TCR, TLR-2, TLR-3, and TLR-4 significantly increased in the GAPDH-S group compared with those in the GAPDH-L and negative control groups. In summary, our results indicate that the optimized recombinant protein GAPDH-S is a promising candidate construct that may be further developed into a multivalent subunit vaccine for strangles.

Mechanism by which the combination of SjCL3 and SjGAPDH protects against Schistosoma japonicum infection.

A vaccine is an important method to control schistosomiasis. Molecules related to lung-stage schistosomulum are considered potential vaccine candidates. We previously showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cathepsin L3 (CL3) displayed differential expression in the lung-stage schistosomula of Schistosoma japonicum cocultured with host cells. In the present study, we prepared the two proteins and detected the protective effects of SjGAPDH by immunizing mice with this protein alone and in combination with SjCL3 with or without Freund's adjuvant. Then, we investigated the possible mechanisms underlying S. japonicum infection. The results showed that vaccination of adjuvanted SjGAPDH decreased the worm burden (37.8%) and egg load (38.1%), and the combination of adjuvanted SjGAPDH and SjCL3 further decreased the worm burden (65.6%) and egg load (70.9%) during Schistosoma japonicum infection. However, the immunization of a combination of adjuvant-free SjGAPDH and SjCL3 displayed a lower protective effect (< 15%) than those of the adjuvanted SjCL3, the adjuvanted SjGAPDH, and a combination of adjuvanted SjGAPDH and SjCL3. Flow cytometric results showed that the frequency of regulatory T cells (Tregs) was lower (P < 0.05) in the group with adjuvanted SjGAPDH and SjCL3 (2.61%) than the remaining groups. The enzyme-linked immunosorbent assay (ELISA) results indicated that except for the uninfected and infected control groups, the remaining groups displayed a Th1-type shift in immune responses. These results showed the immunization of SjGAPDH resulted in partial protection (approximately 38%); inoculation with a combination of SjCL3 and SjGAPDH in Freund's adjuvant resulted in a high immunoprotective effect (> 65%) against Schistosoma japonicum infection in mice, which was possibly caused by the reduced percentage of Tregs and a Th1-type shift in immune responses; and SjCL3 has no adjuvant-like effect, dissimilar to SmCL3.

Protective immune responses against Schistosoma mansoni infection by immunization with functionally active gut-derived cysteine peptidases alone and in combination with glyceraldehyde 3-phosphate dehydrogenase.

BACKGROUND: Schistosomiasis, a severe disease caused by parasites of the genus Schistosoma, is prevalent in 74 countries, affecting more than 250 million people, particularly children. We have previously shown that the Schistosoma mansoni gut-derived cysteine peptidase, cathepsin B1 (SmCB1), administered without adjuvant, elicits protection (>60%) against challenge infection of S. mansoni or S. haematobium in outbred, CD-1 mice. Here we compare the immunogenicity and protective potential of another gut-derived cysteine peptidase, S. mansoni cathepsin L3 (SmCL3), alone, and in combination with SmCB1. We also examined whether protective responses could be boosted by including a third non-peptidase schistosome secreted molecule, glyceraldehyde 3-phosphate dehydrogenase (SG3PDH), with the two peptidases. METHODOLOGY/PRINCIPAL FINDINGS: While adjuvant-free SmCB1 and SmCL3 induced type 2 polarized responses in CD-1 outbred mice those elicited by SmCL3 were far weaker than those induced by SmCB1. Nevertheless, both cysteine peptidases evoked highly significant (P < 0.005) reduction in challenge worm burden (54-65%) as well as worm egg counts and viability. A combination of SmCL3 and SmCB1 did not induce significantly stronger immune responses or higher protection than that achieved using each peptidase alone. However, when the two peptidases were combined with SG3PDH the levels of protection against challenge S. mansoni infection reached 70-76% and were accompanied by highly significant (P < 0.005) decreases in worm egg counts and viability. Similarly, high levels of protection were achieved in hamsters immunized with the cysteine peptidase/SG3PDH-based vaccine. CONCLUSIONS/SIGNIFICANCE: Gut-derived cysteine peptidases are highly protective against schistosome challenge infection when administered subcutaneously without adjuvant to outbred CD-1 mice and hamsters, and can also act to enhance the efficacy of other schistosome antigens, such as SG3PDH. This cysteine peptidase-based vaccine should now be advanced to experiments in non-human primates and, if shown promise, progressed to Phase 1 safety trials in humans.

Papain-Based Vaccination Modulates Schistosoma mansoni Infection-Induced Cytokine Signals.

We have previously shown that immunization of outbred rodents with cysteine peptidases-based vaccine elicited type 2-biased immune responses associated with consistent and reproducible protection against challenge Schistosoma mansoni. We herein start to elucidate the molecular basis of C57BL/6 mouse resistance to S. mansoni following treatment with the cysteine peptidase, papain. We evaluated the early cytokine signals delivered by epidermal, dermal, and draining lymph node cells of naïve, and S. mansoni -infected mice treated 1 day earlier with 0 or 50 µg papain, or immunized twice with papain only (10 µg/mouse), papain-free recombinant S. mansoni glyceraldehyde 3-phosphate dehydrogenase and 2-Cys peroxiredoxin peptide (10 and 15 µg/mouse, respectively = antigen Mix), or papain-adjuvanted antigen Mix. Schistosoma mansoni infection induced epidermal and lymph node cells to release type 1, type 2 and type 17 cytokines, known to counteract each other. Expectedly, humoral immune responses were negligible until patency. Papain pretreatment or papain-based vaccination diminished or shut off S. mansoni infection early induction of type 1, type 17 and type 2 cytokines except for thymic stromal lymphopoietin and programmed the immune system towards a polarized type 2 immune milieu, associated with highly significant (P < 0.005 - <0.0001) resistance to S. mansoni infection.

Vaccination with recombinant Mycoplasma bovis GAPDH results in a strong humoral immune response but does not protect feedlot cattle from an experimental challenge with M. bovis.

Mycoplasma bovis continues to cause significant disease in feedlots and dairy farms. The ability of the micro-organism to evade the immune system of the host combined with the lack of effective vaccines makes this disease difficult to control. Bacterin-based vaccines have not been successful in field trials and in some cases enhance the disease. In an attempt to develop a sub-unit vaccine, we used the conserved M. bovis glyceraldehyde-3-phosphate (GAPDH) protein in combination with a protein extract prepared from three M. bovis isolates to immunize feedlot animals. After challenge with a combination of three M. bovis isolates, there were differences in the proportion of weight loss between the control and vaccinated groups but no differences in rectal temperature and survival rate in all the groups. In addition, there were no significant differences between the proportions of lungs lesions in all the groups despite the percentages of lesions being higher in the vaccinated groups. These findings indicate that the M. bovis GAPDH protein is not a suitable antigen for a vaccine against this pathogen.

Application of the mammalian glyceraldehyde-3-phosphate dehydrogenase gene for sample quality control in multiplex PCR for diagnosis of leishmaniasis

Leishmaniasis is a neglected disease endemic in five continents. It is a severe disease that may lead to death, and its early detection is important to avoid severe damage to affected individuals. Molecular methods to detect Leishmania are considered alternatives to overcome the limitations presented by conventional methods. The aim of this study was to develop multiplex PCR systems able to detect small amounts of target DNA of Leishmania infantum and Leishmania braziliensis, and the gene coding for glyceraldehyde-3-phosphate dehydrogenase (G3PD) in mammals, enabling quality evaluation of the sample simultaneously with detection of the specific target. The systems created for G3PD recognition were combined with detection systems for L. infantum and L. braziliensis to compose multiplex PCR systems for visceral (mVL) and cutaneous (mACL) leishmaniasis diagnosis. The multiplex PCR systems developed were assessed in blood samples from five different species of mammal reservoirs involved in the disease cycle in Brazil, and 96 and 52 human samples from patients with suspected visceral leishmaniasis (VL) and cutaneous leishmaniasis (ACL), respectively. Three G3PD detection systems were created (G3PD1, G3PD2 and G3PD3) with different product sizes, G3PD2 was chosen for the formation of multiplex PCR systems. The two multiplex PCR systems (mVL and mACL) were reproducible in all species evaluated. Results of test samples (sensitivity, specificity and efficiency) suggest its use in routine diagnosis, research activities in medicine and veterinary medicine. Additionally, the systems designed to detect the G3PD gene are capable of combining with other targets used for molecular diagnosis of infectious diseases. Concerning leishmaniasis, the multiplex PCR systems can be used in epidemiological studies for the detection of new and classic reservoirs, which may contribute to the reliability of results and development of actions to control the disease.(AU)