Comparison of DC/CFA and alum as adjuvants for induction of immune resopnses to the β-(Man)₃-Fba conjugate vaccine.

<p>Serum samples were collected 14 days after immunization, diluted 1∶100 and tested by ELISA on plates coated with either synthetic Fba-MAP or β-(Man)₃. Immune sera from mice immunized with the β-(Man)₃-Fba DC/CFA showed greater antibody titers to both the Fba peptide (A) and the β-(Man)₃ epitopes (B) than sera from groups that received β-(Man)₃-Fba in alum. (C) A high degree of protection was induced by the β-(Man)₃-Fba pulsed DCs, and slight protection was observed when alum was used as the adjuvant as compared to DPBS, DC+CFA or alum adjuvant unimmunized controls.</p

β-(Man)₃-Fba administered along with either alum or MPL adjuvants induced modest antibody responses and slight protection against disseminated candidiasis.

<p>The β-(Man)₃-Fba conjugate was administered as a mixture with either alum or MPL adjuvants in BALB/c mice. Serum samples were collected 14 days after immunization, diluted 1∶100 and tested by ELISA on plates coated with synthetic β-(Man)₃ or Fba-MAP. After the first booster immunization, immune sera from vaccinated mice showed modest antibody responses to Fba peptide (A) and relatively weak antibody responses to β-(Man)₃ epitope (B) (C) The survival was also slightly extended in mice that received β-(Man)₃-Fba in MPL and slight protection was observed when alum was used as the adjuvant as compared to DPBS or adjuvant unimmunized controls.</p

ELISA titers against microtiter wells coated with synthetic Fba peptide.

<p>ELISA titers against microtiter wells coated with synthetic Fba peptide.</p

ELISA titers against microtiter wells coated with synthetic β-(Man)₃ epitope.

<p>ELISA titers against microtiter wells coated with synthetic β-(Man)₃ epitope.</p

Antibody isotype distribution of responses to Fba and β-(Man)₃.

<p>Antibody isotype distribution of responses to Fba and β-(Man)₃.</p

β-Man)₃-Fba-TT conjugate with or without adjuvant markedly induced high antibody titers and protection against disseminated candidiasis in immunized mice as compared to controls.

<p>Mice immunized with β-(Man)₃-Fba-TT prepared in either alum or MPL, or without adjuvant developed robust antibody responses against both the Fba peptide (A) and the β-(Man)₃ epitope (B). (C) Protective immunity was induced by (β-Man)₃-Fba-TT when either alum or MPL was used as the adjuvant. Protection was nearly as great even when adjuvant was omitted as compared to DPBS or adjuvant only controls (<i>P</i><0.01). (D) Immunized mice had reduced or non-detectable CFUs per kidney pairs compared to control groups (<i>P</i><0.001).</p

Passive transfer experiment was performed to confirm that antibody is responsible for the protection.

<p>(A) As immunized mice, immune sera recipients had a prolonged survival time (<i>P</i><0.01), confirming that induced antibodies were protective. (B) The serum from (β-Man)₃-Fba-TT immunized animals was capable of reducing the fungal load in the mouse kidneys compared with the infectious burden in mice were given DPBS or pre-absorbed sera (<i>P</i><0.001).</p

Vaccination with β-(Man)₃-Fba-TT in either alum or MPL markedly increased both β-(Man)₃ and Fba peptide-specific antibody titers in sensitized mice as compared to controls.

<p>Serum samples were collected 14 days after immunization, diluted 1∶100 and tested by ELISA on plates coated with cell wall mannan or peptide. MAbs B6.1 and E2-9 that are specific for β-(Man)₃ and Fba, respectively, were used as positive controls. Mice immunized with β-(Man)₃-Fba-TT prepared in either alum or MPL induced robust antibody responses against both the Fba peptide (A) and the β-(Man)₃ (B) epitopes. However, mice that received either Fba or Fba-TT in either adjuvant produced weak anti-Fba responses.</p

The (β-Man)₃-TT conjugate vaccine is immunogenic and protective against disseminated candidiasis in outbred mice.

<p>Outbred CFWSwiss Webster (01S60) mice were immunized with the β-(Man)₃-Fba-TT conjugate alone or mixed with adjuvants alum and MPL; control mice were immunized with adjuvants (alum+MPL) only or DPBS buffer. Mice immunized with β-(Man)₃-Fba-TT in either alum+MPL, or without adjuvant induced robust antibody responses against both the β-(Man)₃ epitope (A) and the Fba peptide (B). (C) Protective immunity was induced by (β-Man)₃-Fba-TT with or without adjuvant as noted by their prolonged survival time as compared to control mice that received DPBS or adjuvants alone (<i>P</i><0.01). (D) Immunized mice had reduced or non-detectable CFUs per kidney pairs compared to control groups.</p

Scheme for synthesis of the conjugate vaccine.

<p>The glycoconjugate vaccine (<b>GV</b>) and the peptide vaccine lacking the mannotriose component (<b>PV</b>) were synthesized from the advanced building blocks <b>1</b>–<b>3</b>. The β-1,2 mannotriose derivatized with a triethylene glycol spacer <b>1</b>; the T-cell tetradecameric peptide (Fba) was assembled on a peptide synthesizer and a triethylene glycol tether was introduced at the C terminal end followed by a single lysine residue which was derivatized on its side chain by a thioacetic acid residue, which gave the building blocks <b>2a</b> or <b>2b</b>. Bromoacetate groups were introduced on approximately 20 of the lysine residues present in tetanus toxoid to give <b>3</b>. <b>GV</b> was assembled by reacting <b>1</b> with <b>2a</b> and then conjugating this product with <b>3</b>. <b>PV</b> was prepared by conjugating 2b with 3. A detailed account of this synthesis will be reported elsewhere (Cartmell <i>et al.</i> Carbohydr. Res submitted).</p