Bacterial phospholipases C as vaccine candidate antigens against cystic fibrosis respiratory pathogens: the Mycobacterium abscessus model.

BACKGROUND: Vaccine strategies represent one of the fighting answers against multiresistant bacteria in a number of clinical settings like cystic fibrosis (CF). Mycobacterium abscessus, an emerging CF pathogen, raises difficult therapeutic problems due to its intrinsic antibiotic multiresistance. METHODS: By reverse vaccinology, we identified M. abscessus phospholipase C (MA-PLC) as a potential vaccine target. We deciphered here the protective response generated by vaccination with plasmid DNA encoding the MA-PLC formulated with a tetra functional block copolymer 704, in CF (ΔF508) mice. Protection was tested against aerosolized smooth and rough (hypervirulent) variants of M. abscessus. RESULTS: MA-PLC DNA vaccination (days 0, 21, 42) elicited a strong antibody response. A significant protective effect was obtained against aerosolized M. abscessus (S variant) in ΔF508 mice, but not in wild-type FVB littermates; similar results were observed when: (i) challenging mice with the "hypervirulent" R variant, and; (ii) immunizing mice with purified MA-PLC protein. High IgG titers against MA-PLC protein were measured in CF patients with M. abscessus infection; interestingly, significant titers were also detected in CF patients positive for Pseudomonas aeruginosa versus P. aeruginosa-negative controls. CONCLUSIONS: MA-PLC DNA- and PLC protein-vaccinated mice cleared more rapidly M. abscessus than ß-galactosidase DNA- or PBS- vaccinated mice in the context of CF. PLCs could constitute interesting vaccine targets against common PLC-producing CF pathogens like P. aeruginosa.

AFP-specific immunotherapy impairs growth of autochthonous hepatocellular carcinoma in mice.

BACKGROUND AND AIMS: In this study, we have assessed the potential of antigen-specific immunotherapy against hepatocellular carcinoma (HCC) in conditions of low tumour burden, in an autochthonous HCC model. METHODS: Diethylnitrosamine (DEN) injected into infant mice results in the development of multi-nodular HCC in which alpha-fetoprotein (AFP) is re-expressed. DEN-injected animals received an antigen-specific immunization with a synthetic vector consisting of a low dose of AFP-encoding plasmid formulated with the amphiphilic block copolymer 704 (DNAmAFP/704). Animals were treated at 4 and 5 months, before macroscopic nodules were detected, and were sacrificed at 8 months. The tumour burden, as well as liver histology, was assessed. AFP and MHC class I molecule expression in the nodules were monitored by qRT-PCR. RESULTS: The AFP-specific immunotherapy led to a significant (65%) reduction in tumour size. The reduced expression of AFP and MHC class I molecules was measured in the remaining nodules taken from the DNAmAFP/704-treated group. CONCLUSIONS: This is the first study demonstrating the relevance of antigen-specific immunotherapy in an autochthonous HCC model. In this context, we validated the use of an anti-tumour immunotherapy based on vaccination with nanoparticles consisting of low dose antigen-encoding DNA formulated with a block copolymer. Our results demonstrate the potential of this strategy as adjuvant immunotherapy to reduce the recurrence risk after local treatment of HCC patients.

DNA/amphiphilic block copolymer nanospheres promote low-dose DNA vaccination.

Intramuscular (i.m.) DNA vaccination induces strong cellular immune responses in the mouse, but only at DNA doses that cannot be achieved in humans. Because antigen expression is weak after naked DNA injection, we screened five nonionic block copolymers of poly(ethyleneoxide)-poly(propyleneoxide) (PEO-PPO) for their ability to enhance DNA vaccination using a beta-galactosidase (betaGal) encoding plasmid, pCMV-betaGal, as immunogen. At a high DNA dose, formulation with the tetrafunctional block copolymers 304 (molecular weight [MW] 1,650) and 704 (MW 5,500) and the triblock copolymer Lutrol (MW 8,600) increased betaGal-specific interferon-gamma enzyme-linked immunosorbent spot (ELISPOT) responses 2-2.5-fold. More importantly, 704 allowed significant reductions in the dose of antigen-encoding plasmid. A single injection of 2 microg pCMV-betaGal with 704 gave humoral and ELISPOT responses equivalent to those obtained with 100 microg naked DNA and conferred protection in tumor vaccination models. However, 704 had no adjuvant properties for betaGal protein, and immune responses were only elicited by low doses of pCMV-betaGal formulated with 704 if noncoding carrier DNA was added to maintain total DNA dose at 20 microg. Overall, these results show that formulation with 704 and carrier DNA can reduce the dose of antigen-encoding plasmid by at least 50-fold.