Synthesis, refolding and protective immune responses of a potential antigen for human Respiratory Syncytial Virus vaccine.

The design of new antigens with both high immunogenic and safety properties is of particular interest to vaccine against infectious diseases. In the present study, we describe the synthesis and the refolding of peptide G20 derived from the Human Respiratory Syncytial Virus (hRSV) G-protein. G20 (MEF G140-190 G144-158) is a peptide of 69 amino acids with two disulfide bridges, which comprises multiple protective B-cell epitopes. It was deleted of the T helper cell epitope 184-198 of the RSV G-protein, which was found to induce pulmonary pathology after RSV challenge in mice. Interestingly, we showed in the present study that G20 generated a highly protective antibody response against RSV challenge in Balb/c mice. Therefore, G20 represents a new potential antigen for an RSV vaccine.

Stability and CTL activity of N-terminal glutamic acid containing peptides.

Several cytotoxic T lymphocyte peptide-based vaccines against hepatitis B, human immunodeficiency virus and melanoma were recently studied in clinical trials. One interesting melanoma vaccine candidate alone or in combination with other tumor antigens, is the decapeptide ELA. This peptide is a Melan-A/MART-1 antigen immunodominant peptide analog, with an N-terminal glutamic acid. It has been reported that the amino group and gamma-carboxylic group of glutamic acids, as well as the amino group and gamma-carboxamide group of glutamines, condense easily to form pyroglutamic derivatives. To overcome this stability problem, several peptides of pharmaceutical interest have been developed with a pyroglutamic acid instead of N-terminal glutamine or glutamic acid, without loss of pharmacological properties. Unfortunately compared with ELA, the pyroglutamic acid derivative (PyrELA) and also the N-terminal acetyl-capped derivative (AcELA) failed to elicit cytotoxic T lymphocyte (CTL) activity. Despite the apparent minor modifications introduced in PyrELA and AcELA, these two derivatives probably have lower affinity than ELA for the specific class I major histocompatibility complex. Consequently, in order to conserve full activity of ELA, the formation of PyrELA must be avoided. Furthermore, this stability problem is worse in the case of clinical grade ELA, produced as an acetate salt, like most of the pharmaceutical grade peptides. We report here that the hydrochloride salt, shows higher stability than the acetate salt and may be suitable for use in man. Similar stability data were also obtained for MAGE-3, another N-terminal glutamic acid containing CTL peptide in clinical development, leading us to suggest that all N-terminal glutamic acid and probably glutamine-containing CTL peptide epitopes may be stabilized as hydrochloride salts.

Lipophilic quaternary ammonium salt acts as a mucosal adjuvant when co-administered by the nasal route with vaccine antigens.

Nasal administration of vaccines is an attractive approach which offers several significant advantages over traditional intramuscular vaccine delivery. These advantages include easier administration and induction of immune responses in the mucosal secretions of the body. In this study we describe a new potent nasal adjuvant, dimethyldioctadecylammonium bromide (DDA), that induces both mucosal and systemic immune responses when co-administered with diphtheria toxoid (DT), tetanus toxoid (TT) and BBG2Na antigens. In particular, we show that the nasal delivery of recombinant fragment (BBG2Na) of the G protein of respiratory syncytial virus (RSV) mixed with DDA induces both local and systemic anti-RSV immune responses and protects against viral challenge. Furthermore, we provide evidence that the DDA+BBG2Na vaccine does not induce lung immunopathology upon subsequent RSV challenge.

Stability and CTL-activity of P40/ELA melanoma vaccine candidate.

The decapeptide ELA (ELAGIGILTV), a Melan-A/MART-1 antigen immunodominant peptide analogue, is an interesting melanoma vaccine candidate alone or in combination with other tumour antigens. P40, the recombinant outer membrane protein A of Klebsiella pneumoniae (kpOmpA), was recently shown to target dendritic cells and to induce peptide-specific CTLs. Here we investigated the adjuvant role of P40 mixed or chemically conjugated to ELA. This compound is an N-terminal glutamic acid-containing peptide. However, it has been reported that the amino group and the gamma-carboxylic group of glutamic acids easily condense to form pyroglutamic derivatives. Usually, to overcome this stability problem, peptides of pharmaceutical interest were developed with a pyroglutamic acid instead of N-terminal glutamic acid, without loss of pharmacological properties. Unfortunately, the pyroglutamic acid derivative (PyrELA) as well as the N-terminal acetyl capped derivative (AcELA) failed to elicit CTL activity when mixed with P40 adjuvant protein. Despite the apparent minor modifications introduced by PyrELA and AcELA, these two derivatives have probably lower affinity than ELA for the class I Major Histocompatibility Complex. Furthermore, this stability problem is worse in the case of clinical grade ELA, produced as an acetate salt, like most of the pharmaceutical grade peptides. We report here that the hydrochloride shows a higher stability than the acetate and may be suitable for use in man.

Synthesis and characterization of Respiratory Syncytial Virus protein G related peptides containing two disulfide bridges.

Respiratory Syncytial Virus (RSV) is the most important cause of bronchiolitis and viral pneumonia in infants and young children. Approximately 100000 children are hospitalized in the USA each year as a result of RSV infections. During the research and development of subunit human Respiratory Syncytial Virus vaccines (hRSV), we have produced numerous synthetic peptides and recombinant proteins containing the four cysteines of the highly conserved central region of the G attachment protein. For several of these disulfide-containing peptides, all possible oxidized isomers were synthesized using various oxidation conditions and resulting in different ratios of isomers. Each isolated isomer was fully characterized by RP-HPLC, FZCE and ES-MS after purification by preparative RP-HPLC. The different cysteine pairings were unambiguously established after enzymatic digestion, LC-MS analysis and peptide microsequencing. These synthesis and analytical methods were developed for the characterization on one hand, of recombinant fusion protein BBG2Na which is currently being investigated in advanced clinical phases as a very promising vaccine candidate, and on the other hand, for peptides which were synthesized to be evaluated as conjugate vaccines or as immunochemical tools, after covalent coupling to carrier proteins. Furthermore, these studies allowed us to determine which of the different possible isomers was the most stable and probably the preferred form in native conditions. Finally, the different oxidation and analysis conditions, should be useful for disulfide pairing studies of other peptides and proteins having the same 'xCxxCxxxxxCxxxCx' framework, such as G proteins of non-human RSV strains, developed by other groups as veterinary vaccine candidates for example.

Synthesis and characterization of respiratory syncytial virus protein G related peptides containing two disulphide bridges.

Respiratory Syncytial Virus (RSV) is the most important cause of bronchiolitis and viral pneumoniae in infants and young children. Approximately 100,000 children are hospitalized in the USA each year as a result of RSV infections. During the research and development of subunit human RSV vaccines, we have produced numerous synthetic peptides and recombinant proteins containing the four cysteines of the highly conserved central region of the G attachment protein. For several of these disulphide bridges containing peptides, all possible oxidizing isomers were synthesized using various oxidising conditions, resulting in different ratios of isomers. Each isolated isomer was fully characterized by RP-HPLC, FZCE and ES-MS after purification by preparative RP-HPLC. The different cysteine pairings were unambiguously established after enzymatic digestion, LC-MS analysis and peptide microsequencing. These synthetic and analytical methods were developed for the characterization of recombinant fusion protein BBG2Na which is currently investigated in clinical phase II and seems to be as a very promising vaccine candidate, and for peptides which were synthesized to be evaluated as conjugate vaccines or as immunochemical tools, after covalent coupling to carrier proteins. Furthermore, these studies allowed us to determine which of the different possible isomers was the most stable and probably the preferred form in native conditions. Finally, the different oxidising and analysis conditions, should be useful for disulphide pairing studies of other peptides and proteins having the same "xCxxCxxxxxCxxxCx" framework, such as G proteins of non-human RSV strains, developed for example as veterinary vaccine candidates.