Vaccination with Met6 peptide induced antibody production and protection against disseminated candidiasis in mice. C. albicans
cell surface peptides were selected from previously identified cell wall proteins that are expressed during pathogenesis
of human disseminated candidiasis [10
]. Among the six peptide epitopes, we first focused on peptide Fba, which turned out to be an excellent peptide vaccine against C. ablicans
]. When used alone to pulse DCs for subsequent immunizations, peptide Fba induced robust protective responses, which was medicated by Fba peptide-specific antibodies [6
]. By the same antigen-pulsed DC-based immunization strategy favoring production of antibodies as described before [5
], another cell surface peptide Met6 also induced good protection as evidenced by survival (Figure 1A) and reduced or non-detectable fungal burden (colony forming units, CFUs) in kidney (Figure 1B), a target organ in disseminated candidiasis, in mice challenged with the fungus. Antibody activity specific for the Met6 peptide appears to be responsible for the anti-Candida
protection, as was demonstrated by experiments involving passive transfer of whole immune serum from Met6 peptide vaccinated mice, which conferred protection to naïve mice (Figure 1C). Such protection was not conferred by control DPBS buffer and the protection was abrogated when immune
serum was pre-absorbed by fungal cells prior to the passive transfer protection test (Figure 1C).
Vaccination double peptide mixture didn’t improve antibody production and protection against disseminated candidiasis in mice. To induce more effective protection, the peptide Fba was mixed with peptide Met6 to form double-peptide mixture for immunization in mice. By the similar dendritic cell (DC)-based approach, we tested each individual peptide, Met6 and Fba, side by side with peptide mixture for vaccine efficacy. Immunized groups that received the peptide Fba, peptide Met6 or Fba-Met6 mixture vaccines showed 60-80% survival throughout the 50 days post-challenge observation period and survived significantly longer as compared to DPBS or DC only controls following the lethal challenge (Figure 2A). Peptide or peptide mixture immunized groups had greatly reduced or even non-detectable viable fungal CFUs in kidneys (Figure 2B) as compared to control animals (p<0.0001). However, The Fba/Met6 peptide mixture didn’t increase protective efficacy as compared to that induced by each individual peptide vaccine alone (Figure 1A), and Fba/Met6 mixture groups didn’t show significantly reduced CFUs in kidneys
as compared to individual peptide groups (Figure 2B). There is no significant difference in peptide-related antibody responses between Fba/Met6 mixture group and each individual peptide group (data not shown).
Peptides Fba-8MAP and Met6-8MAP administered along with alum induced only modest protection against candidiasis. By use of the powerful DC-based immunization approach to overcome the weak immune responses of small molecules, the small synthetic peptide Fba and peptide Met6 vaccines were able to provide protection against disseminated candidiasis in mice [12
]. However, the immunization approach could be inappropriate for human use, due to the complicated procedure and potentially expensive cost. To resolve this problem, Fba and Met6 peptide was each conjugated
to multiple antigenic peptide (MAP) system, of which the lysine core displayed approximately eight copies of the peptide epitope, for immunization. To move toward a peptide vaccine against disseminated candidiasis more acceptable for human use, Fba-8MAP and Met6-8MAP each was administered as a mixture with alum (aluminum hydroxide gel, Sigma). Mice were immunized by subcutaneous (s.c.) injection of 0.1 ml containing 2.5 µg of Fba-8MAP or Met6-8MAP mixed with 50 µg alum on days 1, 21 and 42. Negative control groups of mice were given a same volume of DPBS buffer or alum adjuvant only. Groups immunized with peptide pulsed DCs were used as positive controls. Serum samples were collected 14 days after immunization, diluted 1:200 and tested by ELISA on plates coated with synthetic peptide-8MAP. After the first booster, immune sera from mice immunized with Fba or Met6 peptide prepared in alum showed that antibody responses to each preparation peptide were about 4-5 fold greater over background sera obtained from mice that received DPBS or adjuvant only. However, the peptide specific antibody responses were relatively weaker as compared to that induced by peptide pulsed DC immunization approach (Figure 3A). Following the second booster immunization, an isotype switch from IgM to IgG of Fba and Met6 specific antibodies were observed in the sera from both peptide-DC and peptide-8MAP immunized mice (data not shown), which suggested induction of an immune memory response. After challenge with a lethal dose of live C. albicans
cells (3153A), the groups vaccinated with Fba-8MAP or Met6-8MAP mixed with alum had prolonged
survival times as compared to two control groups; however the protection was not as strong as that induced by the peptide pulsed DC approach. Mice immunized with Fba-8MAP or Met6-8MAP administered along with alum had only 40% survival, while the groups immunized with peptide pulsed DC had better survival- 60-80% up to 50 days (Figure 3B). As expected, along with prolonged survival times, all the immunized groups had reduced live fungal cells in their kidneys as compared to controls. Fba-DC and Met6-DC groups had the least CFUs in kidneys among all the vaccinated groups (Figure 3C).
Double chimeric peptide pulsed DC vaccination induced solid protection against disseminated candidiasis in mice. As compared to single peptide vaccine, double peptide epitope vaccine may have more advantages, such as increased potency and breadth in protection. Since Fba and Met6 double-peptide mixture didn’t induce better protection as compared to that induced by each individual peptide, we then used a different strategy by conjugating two peptides into one chimeric vaccine. Fba peptide was conjugated to the N-terminus of Met6 peptide through double lysine linker (-KK-), which is the target sequence of the lyzosomal protease cathepsin B for antigen processing in the context of MHC-II antigen presentation [13
], to form Fba-Met6 double chimeric peptide vaccine. Since Fba peptide consistently induced best protection by DC-based immunization, mice immunized with Fba-DC were used as positive control to compare the efficacy between Fba peptide vaccine and Fba- Met6 chimeric peptide
vaccine. After animals were challenged, similar protection patterns were observed in all the immunized mice (Figure 4A).
In addition to prolonged survival times, each immunized group had reduced or non-detectable CFUs in their kidneys as compared to non- immune mice (Figure 4B). Impressively, Fba-Met6 chimeric vaccine induced 100% complete protection up to 60 days when the experiment was terminated
(Figure 4A), and only one mouse in that group had detectable low CFU in her kidneys (Figure 4B). Our data indicate Fba-Met6 double chimeric peptide vaccine performed in a superior fashion as compared to other individual peptides or peptide mixtures. We also tested all the related peptide vaccines in C57BL/6 mice, which are more prone to Th1 responses, and obtained similar results (data not shown).
Combined IgG3 MAb (M2-4) and IgM MAb (E2-9) conferred enhanced protection against systemic candidiasis in passive transfer experiments. A monoclonal antibody (MAb IgG3 M2-4) specific for Met6 peptide was obtained by use of standard hybridoma techniques. Development of monoclonal antibodies specific for Met6 peptide affords us not only with important probes
to study fungal expression of the peptide, but also provides us with the possibility of producing an unlimited supply of protective antibody for in vivo applications. First, MAb M2-4 was detected by an indirect immunofluorescence antibody test to confirm its specific reactivity with Met6 peptide on the cell surface of C. albicans
(data not shown), and then BALB/c mice were given an i.p dose of MAb M2-4 four hours before hematogensous challenge with a lethal dose of C. albicans
3153A. Protective IgM MAb E2-9 specific for Fba peptide, which was isolated by us before , was used as positive control. Mice received either MAb M2-4 or MAb E2-9 had prolonged survival as compared to control animals that received either DPBS or adsorbed MAbs (Figure 5A); surprisingly, the MAbs combination (M2-4/E2-9) treatment was able to provide the best protection, to protect the naive mice completely (100% survival). Consistently, the group received treatment of two protective MAb in combination had the least CFUs in kidneys among all the groups (Figure 5B). To test how many doses of each MAb are needed for the complete protection in naïve mice, each MAb was further evaluated by giving to naïve mice every other day post challenge for two weeks. Interestingly, when either MAb M2- 4 or E2-9 was given once, either MAb was able to provide 60% protection of recipient animals; however, when each MAb was given every other day for two weeks, MAb M2-4 was able to increase protection from 60% to 80%, and E2-9, from 60% to 100%. Two-week treatment with MAb E2-9 eventually reached the same protection provided by giving two-MAb combination once. Obviously, combination of two protective MAbs provides the best protection with reduced dose of each MAb administered. In all the transfer experiments, passive protection was prevented by removal of the MAbs through absorption with C. albicans cells before transfer (data not shown), which provided strong additional evidence for the protection being due to the MAbs.