Immunization with recombinant enolase ofSporothrixspp (rSsEno) confers effective protection against sporotrichosis in mice

Abstract

13In recent years, research has focused on the immunoreactive components of the S. schenckii cell 14 wall that can be relevant targets for preventive and therapeutic vaccines against sporotrichosis, an 15 emergent worldwide mycosis. In previous studies, we identified a 47-kDa enolase as an 16 immunodominant antigen in mice vaccinated with purified fungal wall proteins and adjuvants. In 17 this study, the immunolocalization of this immunogen in the cell wall of S. schenckii and S. 18 brasiliensis is shown for the first time. In addition, a recombinant enolase of Sporothrix spp 19 (rSsEno) was studied with the adjuvant Montanide Pet-GelA (PGA) as a vaccine candidate. The 20 rSsEno was produced with high purity. In addition, mice immunized with rSsEno plus PGA 21 showed increased antibody titers against enolase and increased median survival time compared to 22 nonimmunized or rSsEno-immunized mice. Enolase immunization induced a predominant T-23 helper-1 (Th1) cytokine pattern in splenic cells after in vitro stimulation with rSsEno. Elevated 24 production of interferon-ɣ (IFN-ɣ) and interleukin-2 (IL-2) was observed with other cytokines 25 involved in the innate immune defense, such as TNF-alpha, IL-6, and IL-4, which are necessary 26 for antibody production. These results suggest that we should continue testing this antigen as a 27 potential vaccine candidate against sporotrichosis. 28 29 Introduction 30 Sporotrichosis is a subcutaneous mycosis of subacute or chronic evolution caused by traumatic 31 inoculation or the inhalation of spores of different species of the Sporothrix genus affecting both 32 humans and animals 1 . The disease has a universal geographical distribution, although it is endemic 33 in Latin America, including in Peru, México, Colombia, Guatemala and, especially, Brazil, where 34 in the last 20 years, it became an important zoonosis, with the cat being the main source of 35 transmission 2-4 . Species of the Sporothrix genus are thermodymorphic fungi with a saprophytic 36 life at 25 °C and a filamentous form. The parasitic form at 35-37 °C is a yeast 1,5 . The human 37 infection is acquired in two ways: traumatic inoculation through the skin with materials 38 contaminated with Sporothrix spp or inhalation. Zoonotic transmission principally occurs from 39 cats to humans 6 . 40 The genus Sporothrix is currently classified into two clades: i) the clinical clade, which includes 41 S. brasiliensis, S. globosa, S. luriei and S. schenckii sensu stricto and ii) the environmental clade, 42 composed mainly of species less pathogenic to man and animals, such as S. mexicana, S. pallida 43 and S. chilensis 7,8 . Brazil is the only country that has reported all species of the clinical clade, and 44 S. brasiliensis is the most virulent species 9,10 . This species is also the most prevalent during 45 zoonotic transmission through deep scratches and bites from infected cats 8 . In this country, though 46 sporotrichosis has been reported in most states, the disease is a neglected disease, particularly in 47 ...

A Recombinant Enolase-Montanide™ PetGel A Vaccine Promotes a Protective Th1 Immune Response against a Highly Virulent Sporothrix schenckii by Toluene Exposure

“…In other recent work, we developed recombinant enolase that was a key antigen in SsCWPs and it was evaluated in a vaccine formulated with PGA as an adjuvant. Again, protective immune response in vaccinated mice after a challenge with S. brasiliensis was observed with a survival rate above 90% after 45 days of intravenous fungal challenge [22].…”

“…Before the evaluation of the protective effect of vaccination, the immunogenicity of the vaccine after two subcutaneous doses of PGA+rSsEno was evaluated. In the aforementioned work [22], we evaluated the immunogenicity and efficacy of this vaccine candidate using three subcutaneous administrations and PGA 10%. However, here the immunogenicity of the vaccine was evaluated after two doses and PGA 5%, as we used in another vaccine candidate with SsCWPs [21].…”

“…The detailed procedures were previously described [22]. Briefly, the gene that encodes S. schenckii enolase with molecular mass 47 kDa and 438 amino acids (access code: ERS97971.1, GenBank database) was synthesized by Epoch Life Science Inc. (Missouri, TX, USA).…”

“…rSsEno IgG antibody titration was conducted as described previously [22]. Briefly, a 96-well ELISA plate (Merck KGaA Darmstadt, Germany) was coated with 5 μg rSsEno/mL in PBS and at 4 °C (overnight).…”

“…However, AH stimulated the development of granulomas in the inoculation site while PGA-based vaccine exhibited a Th1 protective response and better local tolerance than AH-based vaccine in vaccinated mice [21]. In other recent study, we evaluated the immunogenic and protective effect of enolase, one of the main antigens that were found in SsCWPs, against S. brasilensis , the most virulent species of the genus Sporothrix [22]. The recombinant enolase of S. schenckii (SsEno) was formulated with PGA and after three subcutaneous administrations in mice, a significant specific immune response was observed.…”