HisAK70: progress towards a vaccine against different forms of leishmaniosis

Abstract

Background: Leishmania major and Leishmania infantum are among the main species that are responsible for cutaneous leishmaniosis (CL) and visceral leishmaniosis (VL), respectively. The leishmanioses represent the second-largest parasitic killer in the world after malaria. Recently, we succeeded in generating a plasmid DNA (pCMV-HISA70m2A) and demonstrated that immunized mice were protected against L. major challenge. The efficacy of the DNA-vaccine was further enhanced by the inclusion of KMP-11 antigen into the antibiotic-free plasmid pVAX1-asd.

Methods: Here, we describe the use of a HisAK70 DNA-vaccine encoding seven Leishmania genes (H2A, H2B, H3, H4, A2, KMP11 and HSP70) for vaccination of mice to assess the induction of a resistant phenotype against VL and CL.

Results: HisAK70 was successful in vaccinated mice, resulting in a high amount of efficient sterile hepatic granulomas associated with a hepatic parasite burden fully resolved in the VL model; and resulting in 100% inhibition of parasite visceralization in the CL model.

Conclusions: The results suggest that immunization with the HisAK70 DNA-vaccine may provide a rapid, suitable, and efficient vaccination strategy to confer cross-protective immunity against VL and CL.

Fig. 1

Schematic diagram of the construction of pVAX::HISAK70-asd and pVAX-asd. The kanamycin resistance gene in the eukaryotic expression vectors was replaced by the asd gene. a To generate pVAX::HISAK70 without kanamycin resistance cassette (Kan^r), inverse PCR with primers noKanPacI-1R 5’-CTTGTTTAATTAA GCGAAACGATCCTCATCCTGTC-3’ (PacI site underlined) and noKanKpnI-1D 5’-GACGAGGGTACCATTATTAACGCTTACAATTTC-3’ (KpnI site underlined) were employed for outward amplification. The asd gene starting at bp 170 and ending at bp 1345 was PCR amplified from bacterial chromosomal with primers asdKpnI-1D 5’ CTGCAAGGTACCCTACGCCAACTGGCGCAGCAT-3’ (KpnI site underlined) and the asdPacI-1R 5’-TTGGCTGTTAATTAAATGGTGAAGGATGCGCCACAG-3’ (PacI site underlined) creating an amplicon with PacI and KpnI sites on its ends. b To generate pVAX1 without Kan^r, inverse PCR with primers noKanPacI-1R and noKanKpnI-1D were employed for outward amplification. c–d The PCR products of asd, ∆kan^rpVAX1::HISAK70 and ∆kan^rpVAX1 were each double digested with PacI and KpnI, gel isolated, ligated, and transformed. Thus, pVAX1::HisAK70-asd and pVAX1-asd without resistance gene were obtained

Fig. 2

Expression of HisAK70 in CHO-K1 cells after transfection. a-b HisAK70 protein expression was detected from CHO-K1 transfected cells using specific anti-A2 and anti-E Tag antibodies. Lane 1, total protein extract from empty vector-transfected cells (pVAX1-asd); lane 2, cells transfected with pVAX1::HISAK70-asd; and lane 3, extract from non-transfected CHO-K1 cells. Proteins were separated on a 10 % SDS-PAGE gel. The positions of molecular mass markers are indicated on the right. HisAK70 (152 kDa) and the control protein loading, β-Tubulin (52 kDa), are highlighted by arrows, on the left. Data of one representative out of three independents are given. Relevant portions of each blot are shown

Fig. 3

HisAK70 vaccination induces protection against L. infantum or L. major challenges in mice. a Parasite burden was assessed in the spleen and liver at days 28, 42 and 91 after L. infantum infection by limiting the dilution assay. b Course of L. major infection in mice. The mean diameter of induration (± S.D) in the footpad at various times after infection. c Mean number of parasites per popliteal DLN and spleen (± S.D) at 5 weeks after L. major infection. Data are presented as the mean ± S.D. (n = 5). P.N.D., parasites not detected. Asterisks indicate P < 0.05 with respect to control mice

Fig. 4

HisAK70 vaccinated mice develop efficient sterile granulomas. Percentage of hepatic granuloma maturation and representative granulomas from H&E stained liver sections at day 42 p.i. in (a) PBS, (b) empty vector and (c) HisAK70 vaccinated mice. Control mice show high amount of immature granulomas, whereas HisaK70 vaccinated mice show well-developed mature and sterile granulomas. Images were acquired at the indicated magnifications and arrows indicate the presence of amastigotes in the granulomas. All data are presented as the representative mean from each experimental group of mice. Asterisks indicate P < 0.01 with respect to control mice. IM, immature granuloma; M, mature granuloma; Sterile, parasite-free granuloma