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. 2018 Mar 15:9:465.
doi: 10.3389/fimmu.2018.00465. eCollection 2018.

New Vaccine Formulations Containing a Modified Version of the Amastigote 2 Antigen and the Non-Virulent Trypanosoma cruzi CL-14 Strain Are Highly Antigenic and Protective against Leishmania infantum Challenge

Ana Paula M M Almeida  1 , Leopoldo F M Machado  1   2 , Daniel Doro  1   2 , Frederico C Nascimento  1 , Leonardo Damasceno  3 , Ricardo Tostes Gazzinelli  2   4 , Ana Paula Fernandes  1 , Caroline Junqueira  2
Affiliations

New Vaccine Formulations Containing a Modified Version of the Amastigote 2 Antigen and the Non-Virulent Trypanosoma cruzi CL-14 Strain Are Highly Antigenic and Protective against Leishmania infantum Challenge

Ana Paula M M Almeida et al. Front Immunol. .

Abstract

Visceral leishmaniasis (VL) is a major public health issue reported as the second illness in mortality among all tropical diseases. Clinical trials have shown that protection against VL is associated with robust T cell responses, especially those producing IFN-γ. The Leishmania amastigote 2 (A2) protein has been repeatedly described as immunogenic and protective against VL in different animal models; it is recognized by human T cells, and it is also commercially available in a vaccine formulation containing saponin against canine VL. Moving toward a more appropriate formulation for human vaccination, here, we tested a new optimized version of the recombinant protein (rA2), designed for Escherichia coli expression, in combination with adjuvants that have been approved for human use. Moreover, aiming at improving the cellular immune response triggered by rA2, we generated a recombinant live vaccine vector using Trypanosoma cruzi CL-14 non-virulent strain, named CL-14 A2. Mice immunized with respective rA2, adsorbed in Alum/CpG B297, a TLR9 agonist recognized by mice and human homologs, or with the recombinant CL-14 A2 parasites through homologous prime-boost protocol, were evaluated for antigen-specific immune responses and protection against Leishmania infantum promastigote challenge. Immunization with the new rA2/Alum/CpG formulations and CL-14 A2 transgenic vectors elicited stronger cellular immune responses than control groups, as shown by increased levels of IFN-γ, conferring protection against L. infantum challenge. Interestingly, the use of the wild-type CL-14 alone was enough to boost immunity and confer protection, confirming the previously reported immunogenic potential of this strain. Together, these results support the success of both the newly designed rA2 antigen and the ability of T. cruzi CL-14 to induce strong T cell-mediated immune responses against VL in animal models when used as a live vaccine vector. In conclusion, the vaccination strategies explored here reveal promising alternatives for the development of new rA2 vaccine formulations to be translated human clinical trials.

Keywords: Leishmania infantum; Trypanosoma cruzi CL-14; amastigote 2; vaccine; visceral leishmaniasis.

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Figures

Figure 1
Figure 1
Recombinant amastigote 2 (A2) confers immunogenicity and protection against Leishmania infantum challenge in BALB/c mice. (A) Schematic representation of the recombinant A2 protein compared with the original A2 sequence present in Leishmania donovani. Boxes represent the C- or N-terminal sequences, while the circles represent the repetitive amino acid sequences. Although the codon optimization was performed, it is not represented here. (B) Coomassie-stained SDS-PAGE of purified rA2 (left panel) and specific reactivity in Western blot with an anti-A2 monoclonal antibody (right panel). (C) Mice immunized s.c. with a homologous prime-boost protocol containing phosphate-buffered saline (PBS; negative control) or 10 µg of rA2 administered along monophosphoryl lipid A (MPLA) or Alum/CpG had their serum obtained 21 days after the last immunization dose. Serum samples were individually analyzed by enzyme-linked immunosorbent assay (ELISA) for total IgG, IgG1, or IgG2a, as previously described. (D) Mice immunized as previously described were sacrificed 21 days after the last immunization, and spleen cells were used for IFN-γ analyses by ELISA. For in vitro restimulation, it was used either rA2 or RPMI medium. (E) Mice immunized as previously described were euthanized 30 days after the challenge with L. infantum, and spleens were used for parasite loads’ determination using qPCR assay. *p < 0.05, **p < 0.01, and ***p < 0.001 analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. Results are expressed as the mean and SEM values of duplicate assays using four to six different animals from each group individually analyzed.
Figure 2
Figure 2
Transgenic CL-14 parasites stably express recombinant amastigote 2 (A2). (A) Agarose gel electrophoresis of PCR products confirming specific amplification of the nucleotide sequences enconding the A2 (963pb) antigen by CL-14 A2 DNA extracts. As a positive control, plasmid DNA containing the nucleotide sequence-encoding rA2 was used (see Supplementary Material for plasmid maps). For negative control, we used untransfected CL-14 DNA extract. (B) Western blot analysis of transgenic CL-14 parasites lysates confirming the expression of 24 kDa A2. We used rA2 as a positive control. Monoclonal anti-A2 was used for detection of A2. (C) Immunofluorescence of fixed untransfected (CL-14 WT) and trasngenic CL-14 A2 parasites. 2 × 105 parasites were washed, fixed, and loaded into a poly-l-lysine-coated glass slide. DAPI shows nuclear staining, and the green channel shows protein localization detected by monoclonal anti-A2 with AlexaFluor 488 anti-mouse IgG as secondary antibody. The glass slides were analyzed in a LSM Zeiss microscope.
Figure 3
Figure 3
Prime/boost immunization with CL-14 amastigote 2 (A2) induces an effective immune response in immunized BALB/c mice. (A) Specific antibody response (total IgG, IgG1, and IgG2a) induced by each immunization protocol was measured 21 days after the last immunization dose using serum samples of immunized mice. (B) Levels of total IgG against the live vaccine vector CL-14 were measured by enzyme-linked immunosorbent assay (ELISA) using serum samples of immunized mice. As a coating antigen, 10 µg of total CL-14 antigen extract was used. Mice immunized as previously described were euthanized 21 days after the last immunization dose. Spleen cells were cultured, and supernatants were used for IFN-γ (C) or IL-10 (D) analyses by ELISA. For in vitro re-stimulation, we used either RPMI or rA2. *p < 0.05, **p < 0.01, and ***p < 0.001 analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. Results are expressed as the mean and SEM values of duplicate assays using four to six different animals from each group individually analyzed.
Figure 4
Figure 4
Prime/boost immunization with CL-14 amastigote 2 (A2) induces protection in immunized BALB/c mice 30 days after the challenge animals were euthanized, and DNAs extracted from the spleen (A) or liver (B) were used for parasite burden using qPCR assay. The reduction percentage of parasite burden, based on the phosphate-buffered saline (PBS) group is shown for each group. *p < 0.05 and ***p < 0.001 analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. Results are expressed as the mean and SEM values of duplicate assays using four to six different animals from each group individually analyzed.
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