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From mouse to man: safety, immunogenicity and efficacy of a candidate leishmaniasis vaccine LEISH-F3+GLA-SE.

Coler RN, Duthie MS, Hofmeyer KA, Guderian J, Jayashankar L, Vergara J, Rolf T, Misquith A, Laurance JD, Raman VS, Bailor HR, Cauwelaert ND, Reed SJ, Vallur A, Favila M, Orr MT, Ashman J, Ghosh P, Mondal D, Reed SG - Clin Transl Immunology (2015)

Bottom Line: Two Leishmania proteins-nucleoside hydrolase and a sterol 24-c-methyltransferase, each of which are protective in animal models of VL when properly adjuvanted- were produced as a single recombinant fusion protein NS (LEISH-F3) for ease of antigen production and broad coverage of a heterogeneous major histocompatibility complex population.Based on the sum of preclinical data, we prepared GMP materials and performed a phase 1 clinical study with LEISH-F3+GLA-SE in healthy, uninfected adults in the United States.The vaccine candidate was shown to be safe and induced a strong antigen-specific immune response, as evidenced by cytokine and immunoglobulin subclass data.

View Article: PubMed Central - PubMed

Affiliation: Infectious Disease Research Institute , Seattle, WA, USA.

ABSTRACT
Key antigens of Leishmania species identified in the context of host responses in Leishmania-exposed individuals from disease-endemic areas were prioritized for the development of a subunit vaccine against visceral leishmaniasis (VL), the most deadly form of leishmaniasis. Two Leishmania proteins-nucleoside hydrolase and a sterol 24-c-methyltransferase, each of which are protective in animal models of VL when properly adjuvanted- were produced as a single recombinant fusion protein NS (LEISH-F3) for ease of antigen production and broad coverage of a heterogeneous major histocompatibility complex population. When formulated with glucopyranosyl lipid A-stable oil-in-water nanoemulsion (GLA-SE), a Toll-like receptor 4 TH1 (T helper 1) promoting nanoemulsion adjuvant, the LEISH-F3 polyprotein induced potent protection against both L. donovani and L. infantum in mice, measured as significant reductions in liver parasite burdens. A robust immune response to each component of the vaccine with polyfunctional CD4 TH1 cell responses characterized by production of antigen-specific interferon-γ, tumor necrosis factor and interleukin-2 (IL-2), and low levels of IL-5 and IL-10 was induced in immunized mice. We also demonstrate that CD4 T cells, but not CD8 T cells, are sufficient for protection against L. donovani infection in immunized mice. Based on the sum of preclinical data, we prepared GMP materials and performed a phase 1 clinical study with LEISH-F3+GLA-SE in healthy, uninfected adults in the United States. The vaccine candidate was shown to be safe and induced a strong antigen-specific immune response, as evidenced by cytokine and immunoglobulin subclass data. These data provide a strong rationale for additional trials in Leishmania-endemic countries in populations vulnerable to VL.

No MeSH data available.


Related in: MedlinePlus

LEISH-F3 construct and characterization. (a) Schematic of LEISH-F3 (NS) fusion protein. (b–e) SDS-PAGE and immunoblot of LEISH-F3 (NS). (b) LEISH-F3 was run in reducing and nonreducing conditions on a 4–20% Tris-glycine gel. (c) Immunoblot of LEISH-F3 with rabbit polyclonal antibody to LEISH-F3; 100 ng each of LEISH-F3, NH and SMT; 27 μg of L. donovani whole-cell lysate (WCL). (d) Immunoblot of 2 μg of LEISH-F3 and HMS174 E. coli lysate with antibody to HMS174 E. coli. (e, f) Immunoblot of LEISH-F3 and its component antigens, NH and SMT, with mouse monoclonal antibody (mAb) against (e) NH or (f) SMT; 100 ng each of LEISH-F3, NH and SMT were loaded.
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fig2: LEISH-F3 construct and characterization. (a) Schematic of LEISH-F3 (NS) fusion protein. (b–e) SDS-PAGE and immunoblot of LEISH-F3 (NS). (b) LEISH-F3 was run in reducing and nonreducing conditions on a 4–20% Tris-glycine gel. (c) Immunoblot of LEISH-F3 with rabbit polyclonal antibody to LEISH-F3; 100 ng each of LEISH-F3, NH and SMT; 27 μg of L. donovani whole-cell lysate (WCL). (d) Immunoblot of 2 μg of LEISH-F3 and HMS174 E. coli lysate with antibody to HMS174 E. coli. (e, f) Immunoblot of LEISH-F3 and its component antigens, NH and SMT, with mouse monoclonal antibody (mAb) against (e) NH or (f) SMT; 100 ng each of LEISH-F3, NH and SMT were loaded.

Mentions: Having demonstrated IgG and cell-mediated responses against NH and SMT in Leishmania-infected individuals, as well as immunogenicity and protection against L. donovani, a straightforward approach was to link in tandem the open reading frames of both genes such that the final construct would result in the generation of a single recombinant antigen, NS/LEISH-F3, comprising both antigens for evaluation as a VL vaccine candidate (Figure 2a). The L. donovani sequence for NH and the L. infantum sequence for SMT were used for the LEISH-F3 construct design. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified, scaled-up fermentation lots of LEISH-F3 revealed minor protein aggregation and a single major band at the expected molecular mass of 75 kDa. A similar band profile was observed for samples in reducing and nonreducing conditions, demonstrating the relative absence of protein aggregates (Figure 2b). The identity of the major band, relative absence of aggregates and large protein fragments were confirmed by immunoblotting with a mouse polyclonal serum raised against LEISH-F3 (Figure 2c). The anti-LEISH-F3 polyclonal antibody recognized multiple epitopes and therefore multiple forms of the proteins. As the higher-molecular-weight species is seen with the SMT protein, it suggests that this component of LEISH-F3 is responsible for the multimerization. Furthermore, the absence of bacterial by-products in the purified LEISH-F3 product was confirmed by immunoblotting with polyclonal antibody to Escherichia coli (Figure 2d). Finally, LEISH-F3 was recognized by monoclonal sera raised against a single epitope on each of the single components, confirming the presence of both NH and SMT within the fusion protein (Figure 2e). Based on high levels of homology in the sequence of the NH and SMT genes across additional Leishmania species (Table 1), it is reasonable to assume that these proteins are expressed and recognized during the infectious process of multiple Leishmania species.


From mouse to man: safety, immunogenicity and efficacy of a candidate leishmaniasis vaccine LEISH-F3+GLA-SE.

Coler RN, Duthie MS, Hofmeyer KA, Guderian J, Jayashankar L, Vergara J, Rolf T, Misquith A, Laurance JD, Raman VS, Bailor HR, Cauwelaert ND, Reed SJ, Vallur A, Favila M, Orr MT, Ashman J, Ghosh P, Mondal D, Reed SG - Clin Transl Immunology (2015)

LEISH-F3 construct and characterization. (a) Schematic of LEISH-F3 (NS) fusion protein. (b–e) SDS-PAGE and immunoblot of LEISH-F3 (NS). (b) LEISH-F3 was run in reducing and nonreducing conditions on a 4–20% Tris-glycine gel. (c) Immunoblot of LEISH-F3 with rabbit polyclonal antibody to LEISH-F3; 100 ng each of LEISH-F3, NH and SMT; 27 μg of L. donovani whole-cell lysate (WCL). (d) Immunoblot of 2 μg of LEISH-F3 and HMS174 E. coli lysate with antibody to HMS174 E. coli. (e, f) Immunoblot of LEISH-F3 and its component antigens, NH and SMT, with mouse monoclonal antibody (mAb) against (e) NH or (f) SMT; 100 ng each of LEISH-F3, NH and SMT were loaded.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4488838&req=5

fig2: LEISH-F3 construct and characterization. (a) Schematic of LEISH-F3 (NS) fusion protein. (b–e) SDS-PAGE and immunoblot of LEISH-F3 (NS). (b) LEISH-F3 was run in reducing and nonreducing conditions on a 4–20% Tris-glycine gel. (c) Immunoblot of LEISH-F3 with rabbit polyclonal antibody to LEISH-F3; 100 ng each of LEISH-F3, NH and SMT; 27 μg of L. donovani whole-cell lysate (WCL). (d) Immunoblot of 2 μg of LEISH-F3 and HMS174 E. coli lysate with antibody to HMS174 E. coli. (e, f) Immunoblot of LEISH-F3 and its component antigens, NH and SMT, with mouse monoclonal antibody (mAb) against (e) NH or (f) SMT; 100 ng each of LEISH-F3, NH and SMT were loaded.
Mentions: Having demonstrated IgG and cell-mediated responses against NH and SMT in Leishmania-infected individuals, as well as immunogenicity and protection against L. donovani, a straightforward approach was to link in tandem the open reading frames of both genes such that the final construct would result in the generation of a single recombinant antigen, NS/LEISH-F3, comprising both antigens for evaluation as a VL vaccine candidate (Figure 2a). The L. donovani sequence for NH and the L. infantum sequence for SMT were used for the LEISH-F3 construct design. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified, scaled-up fermentation lots of LEISH-F3 revealed minor protein aggregation and a single major band at the expected molecular mass of 75 kDa. A similar band profile was observed for samples in reducing and nonreducing conditions, demonstrating the relative absence of protein aggregates (Figure 2b). The identity of the major band, relative absence of aggregates and large protein fragments were confirmed by immunoblotting with a mouse polyclonal serum raised against LEISH-F3 (Figure 2c). The anti-LEISH-F3 polyclonal antibody recognized multiple epitopes and therefore multiple forms of the proteins. As the higher-molecular-weight species is seen with the SMT protein, it suggests that this component of LEISH-F3 is responsible for the multimerization. Furthermore, the absence of bacterial by-products in the purified LEISH-F3 product was confirmed by immunoblotting with polyclonal antibody to Escherichia coli (Figure 2d). Finally, LEISH-F3 was recognized by monoclonal sera raised against a single epitope on each of the single components, confirming the presence of both NH and SMT within the fusion protein (Figure 2e). Based on high levels of homology in the sequence of the NH and SMT genes across additional Leishmania species (Table 1), it is reasonable to assume that these proteins are expressed and recognized during the infectious process of multiple Leishmania species.

Bottom Line: Two Leishmania proteins-nucleoside hydrolase and a sterol 24-c-methyltransferase, each of which are protective in animal models of VL when properly adjuvanted- were produced as a single recombinant fusion protein NS (LEISH-F3) for ease of antigen production and broad coverage of a heterogeneous major histocompatibility complex population.Based on the sum of preclinical data, we prepared GMP materials and performed a phase 1 clinical study with LEISH-F3+GLA-SE in healthy, uninfected adults in the United States.The vaccine candidate was shown to be safe and induced a strong antigen-specific immune response, as evidenced by cytokine and immunoglobulin subclass data.

View Article: PubMed Central - PubMed

Affiliation: Infectious Disease Research Institute , Seattle, WA, USA.

ABSTRACT
Key antigens of Leishmania species identified in the context of host responses in Leishmania-exposed individuals from disease-endemic areas were prioritized for the development of a subunit vaccine against visceral leishmaniasis (VL), the most deadly form of leishmaniasis. Two Leishmania proteins-nucleoside hydrolase and a sterol 24-c-methyltransferase, each of which are protective in animal models of VL when properly adjuvanted- were produced as a single recombinant fusion protein NS (LEISH-F3) for ease of antigen production and broad coverage of a heterogeneous major histocompatibility complex population. When formulated with glucopyranosyl lipid A-stable oil-in-water nanoemulsion (GLA-SE), a Toll-like receptor 4 TH1 (T helper 1) promoting nanoemulsion adjuvant, the LEISH-F3 polyprotein induced potent protection against both L. donovani and L. infantum in mice, measured as significant reductions in liver parasite burdens. A robust immune response to each component of the vaccine with polyfunctional CD4 TH1 cell responses characterized by production of antigen-specific interferon-γ, tumor necrosis factor and interleukin-2 (IL-2), and low levels of IL-5 and IL-10 was induced in immunized mice. We also demonstrate that CD4 T cells, but not CD8 T cells, are sufficient for protection against L. donovani infection in immunized mice. Based on the sum of preclinical data, we prepared GMP materials and performed a phase 1 clinical study with LEISH-F3+GLA-SE in healthy, uninfected adults in the United States. The vaccine candidate was shown to be safe and induced a strong antigen-specific immune response, as evidenced by cytokine and immunoglobulin subclass data. These data provide a strong rationale for additional trials in Leishmania-endemic countries in populations vulnerable to VL.

No MeSH data available.


Related in: MedlinePlus