Recombinant Phage Elicits Protective Immune Response Against Systemic S.globosa Infection In Mouse Model

Sporotrichosis is an acute or chronic subcutaneous mycosis caused by the Sporothrix complex.The global spread of sporotrichosis infection has been increasing in recent years.The Sporothrix complex comprises at least six sibling phylogenetic species,including S.pallida,S.brasiliensis,S.globosa,S.luriei,S.mexicana,and S.schenckii.To our knowledge,S.globosa is the most common pathogenic species in Northeast China,irrespective of clinical symptoms or regions where it is isolated.Severe clinical forms of sporotrichosis mainly occur in immunocompromised patients,especially in patients infected with human immunodeficiency virus(HIV).In addition,disseminated cutaneous sporotrichosis has been reported in immunocompetent individuals.Antifungal drugs are commonly used in clinical practice,but they are associated with considerable severe adverse effects such as vomiting,diarrhea,headache,abdominal pain,hypersensitivity,liver dysfunction,and fungal resistance.Humoral and cellular immunities are the most important host defense mechanisms against fungal infections.Therapeutic monoclonal antibodies against S.schenckii are included in the most active fields of laboratory.A growing number of antifungal vaccine candidates have been assessed for their immunogenicity,safety,and ability to confer protection against fungal pathogens.A previous study on a mouse model revealed that immunization of mice with Sporothrix spp.cell wall proteins or whole cells could elicit protective immune responses against subsequent infection of Sporothrix spp.However,no vaccine against Sporothrix spp.has yet been widely applied in the clinical setting.Gp70 is a 70-k Da glycoprotein that has been described as a major adhesionfactor on the cell surface of Sporothrix.It is a cross-immunogenic protein associated with virulence in fungi that undergoes different post-translational modifications and is present as isoforms and glycoforms in the S.schenckii complex proteomes.Monoclonal antibodies(m Abs)against Gp70 have been demonstrated to induce strong protection against Sporothrix fungi.Epitopes(avyvtsntehnsvvaipiar,gptntvshvffsgdqetvfttvk,tvipgqdatcwvaicpathtafvtdir,kpvqhalltplgldr)on Gp70 were identified by application of epitope-finding algorithm(Gen Script,Antigen Design Tool,Optimum Antigen).Phages displaying peptides can induce humoral and cell-mediated immune responses without the need for an adjuvant.Therefore,phages can be an effective delivery system and be useful for the design of vaccines without compromising their safety or tolerability.In our preliminary experiments,phage displaying the peptide“kpvqhalltplgldr” was found to effectively improve the survival time of the mouse.Therefore,in this study,we aimed to display the peptide kpvqhalltplgldr(referred to as KR)on phages and to study the efficacy of this recombinant phage as a vaccine.The recombinant phage represents a potential novel vaccine candidate without the need for an adjuvant against S.globosa.1.Antibody response against phages displaying the KR peptide in immunized mice.Our study showed that immunization with the recombinant phage displaying the kpvqhalltplgldr peptide(RP)could produce serum antibodies,which can bind to Gp70.Together,our work shows that the RP displaying the kpvqhalltplgldr peptide can serve a function similar to that of Gp70 in the treatment of S.globosa infection and has potential for use as a vaccine.The results of our study indicate that the kpvqhalltplgldr peptide displayed on the phage surface can mimic Gp70 and induce Gp70-specific antibody production in mice,which can in turn bind with Gp70 andtreat the infection.Some studies have claimed that mice inoculated with monoclonal antibodies against Gp70 exhibited significant passive protection.Similar to these findings,our results prove that treatment with anti-RP Ig G conferred significant protection against S.globosa infection.2.Recombinant-phage induced Th1 and Th17 mixed response.We used flow cytometry analysis to test the immune skewing of the T cells.The results revealed that percentages of Th1 cells among mice in the RP group were significantly higher than the corresponding percentage in the control groups(mock and HK-SG groups;p =0.041),while there were no significant differences between the percentages of Th1 cells between the mock and HK-SG groups.The percentages of Th17 cells were significantly higher in the RP,mock,and HK-SG groups than in the control group(p= 0.013),although there were no significant differences in the values among the RP,mock,and HK-SG groups.3.Protection by recombinant phage against systemic S.globosa infection in BALB/c mice.Mice were sacrificed to determine whether immunization with recombinant phage could reduce the fungal load.The CFU was quantified in kidneys7 days after infection,and the results were log10 transformed.The CFU was significantly lower in the organs of recombinant phage-immunized mice than those that received phosphate-buffered saline(PBS;).Regarding clearance from the kidney,there were no significant differences between the HK-SG-immunized and recombinant phage-immunized groups.Similar results were observed in the livers.Histopathological examination showed that fewer inflammatory cells were seen in the kidney in the recombinant phage-and HK-SG-immunized mice than in the PBS-and wild-type phage-immunized mice.Also,PBS and wild-type phage-immunized mice showed a greater number of inflammatory cells than recombinant phage andHK-SG-immunized mice.Taken together,these findings revealed that protective immunity to S.globose could be induced by immunization with recombinant phage and HK-SG.Similar results were obtained in the replicates.Furthermore,recombinant phage was tested for its ability to protect animals from sublethal disseminated sporotrichosis.The procedure was established by intravenous inoculation of viable yeast cells(0.2 m L,1 × l08 cells/mouse)in BALB/c mice.The survival time of each group(n = 10)over 14 days following sublethal challenge.The highest survival rates(80%)were seen in mice that were immunized with recombinant phage.In contrast,only 20% of the mice injected with PBS survived.For the animals receiving HK-SG,a greater survival rate was observed at 14 days after infection(70% of the mice immunized with HK-SG were still alive).A significant increased survival rate was observed in recombinant phage-immunized mice compared to PBS-injected mice.In conclusion,we further confirmed that the cellular immune response contributes strongly to host defense against S.globosa infection,and antibodies can abrogate establishment and disease development by themselves.The nature of the mechanisms underlying the resolution of infection in mice treated with recombinant phage is a complex issue.Nevertheless,it can be hypothesized that phage-KR can elicit protective antibodies,which could increase the cell-mediated immune response and antibody production.Therefore,phage-KR is a new and safe strategy for the treatment of sporotrichosis.