Targeting and retention of HPV16 E7 to the endoplasmic reticulum enhances immune tumour protection

Abstract

The endoplasmic reticulum (ER) is where the major histocompatibility complex (MHC) class I molecules are loaded with epitopes to cause an immune cellular response. Most of the protein antigens are degraded in the cytoplasm to amino acids and few epitopes reach the ER. Antigen targeting of this organelle by Calreticulin (CRT) fusion avoids this degradation and enhances the immune response. We constructed a recombinant adenovirus to express the E7 antigen with an ER-targeting signal peptide (SP) plus an ER retention signal (KDEL sequence). In cell-culture experiments we demonstrated that this new E7 antigen, SP-E7-KDEL, targeted the ER. Infection of mice with this recombinant adenovirus that expresses SP-E7-KDEL showed interferon induction and tumour-protection response, similar to that provided by an adenovirus expressing the E7 antigen fused to CRT. This work demonstrated that just by adding a SP and the KDEL sequence, antigens can be targeted and retained in the ER with a consequent enhancement of immune response and tumour protection. These results will have significant clinical applications.

DNA vaccine encoding human papillomavirus antigens flanked by a signal peptide and a KDEL sequence induces a potent therapeutic antitumor effect

“…These results are consistent with previous studies showing that the linkage of E6 and E7 to ER chaperoning proteins causes the translocation of these proteins from the nucleus to the ER (14,26,27,29), resulting in an almost null signal in the nucleus.…”

“…In a previous study using mice, we demonstrated that treatment with an adenovirus (Ad SP-E7-KDEL) expressing antigen E7 fused to a CRT signal peptide (SP: MLLPVPLLLGLLGLAAAL) and a retention peptide (KDEL) exerted a protective antitumor effect equal to that obtained following treatment with the same antigen fused to CRT ( 14 ). In the present study, an improved vaccine was constructed that includes the E6 and E7 antigens, which provides a better antitumor effect according to the literature ( 15 – 17 ).…”

Targeting diseases with genetically engineeredLactococcus lactisand its course towards medical translation

“…These experiments nevertheless, were performed employing DNA vaccines to produce the chimeric antigen [14][15][16][17]. Using adenoviral vectors, we have demonstrated that the fusion of peptides commonly found in the endoplasmic reticulum (ER) (e.g., calreticulin) to an antigen, resulted in the retention of the antigen in the ER, improving its presentation to the MHC I [18]. These experiments indicate that enhanced antigen presentation to the MHC I results in a better cellular immune response [17][18][19].…”

“…Using adenoviral vectors, we have demonstrated that the fusion of peptides commonly found in the endoplasmic reticulum (ER) (e.g., calreticulin) to an antigen, resulted in the retention of the antigen in the ER, improving its presentation to the MHC I [18]. These experiments indicate that enhanced antigen presentation to the MHC I results in a better cellular immune response [17][18][19]. Taking in account these results, it is possible to use the same strategy and, for example, fusing calreticulin to antigens expressed by L. lactis in order to increase the immunogenicity.…”

“…Deletion of essential genes, auxotrophic strains [7] Expression systems employed Constitutive expression, inducible systems (e.g., nisin-inducible system [NICE]) [10,22,28] Augmentation of the immune response Intracellular antigen targeting (antigen fusion with viral proteins, antigen-presenting cells binding proteins), sub-cellular antigen retention (e.g., in the rugged endoplasmic reticulum), immunomodulators (interleukins, viral or heat shock proteins) [14][15][16][17][18]21,29] Increasing protein expression Codon optimization for Lactococcus, synthetic promoters, protease deficient strains [30,31] Route of administration Dependent on the immune response most adequate for the targeted disease and the entry route of the causal agent [3] Antigen location in L. lactis Cytoplasmic, extracellular or associated with the cell [11] cases probably represent opportunistic infection of (in this case) L. lactis, due to the weakened general health of the patients [25,26]. To our knowledge no cases of infection by L. lactis have been reported in healthy individuals.…”