Incomplete Freund's adjuvant reduces arginase and enhances Th1 dominance, TLR signaling and CD40 ligand expression in the vaccine site microenvironment.

BACKGROUND: Immunogenicity of cancer vaccines is impacted by adjuvants and schedule, but systematic assessments of their effects have not been performed. Montanide ISA-51, an incomplete Freund's adjuvant (IFA), is used in many vaccine trials, but concerns have been raised about negative effects in murine studies. We found in humans that IFA enhances systemic immune responses and that repeat vaccination at one site (same site vaccination (SSV)) creates tertiary lymphoid structures (TLS) in the vaccine site microenvironment (VSME). We hypothesized that vaccination with peptides+IFA+pICLC or SSV×3 with peptides in IFA would create an immunogenic milieu locally at the VSME, with activated dendritic cells (DC), TLS-associated chemokines and a Th1-dominant VSME. METHODS: Biopsies of the VSME were obtained from participants on two clinical trials who were immunized with multiple melanoma peptides (MELITAC 12.1) in adjuvants comprising IFA and/or the TLR3-agonist pICLC. Biopsies were obtained either a week after one vaccine or a week after SSV×3. Controls included normal skin and skin injected with IFA without peptides. Gene expression analysis was performed by RNAseq. RESULTS: VSME samples were evaluated from 27 patients. One vaccine with peptides in pICLC+IFA enhanced expression of CD80, CD83, CD86 (p<0.01), CD40 and CD40L (p<0.0001) over normal skin; these effects were significantly enhanced for SSV with peptides+IFA. Vaccines containing pICLC increased expression of TBX21 (T-bet) but did not decrease GATA3 over normal skin, whereas SSV with peptides in IFA dramatically enhanced TBX21 and decreased GATA3, with high expression of IFNγ and STAT1. SSV with peptides in IFA also reduced arginase-1 (ARG1) expression and enhanced expression of TLR adapter molecules TICAM-1 (TRIF) and MYD88. Furthermore, SSV with IFA and peptides also enhanced expression of chemokines associated with TLS formation. CONCLUSIONS: These findings suggest that SSV with peptides in IFA enhances CD40L expression by CD4 T cells, supports a Th1 microenvironment, with accumulation of activated and mature DC. Increased expression of TLR adaptor proteins after SSV with peptides in IFA might implicate effects of the skin microbiome. Reduced ARG1 may reflect diminished suppressive myeloid activity in the VSME. TRIAL REGISTRATION NUMBER: (NCT00705640, NCT01585350).

Raising antibodies against epoxyscillirosidine, the toxic principle contained in Moraea pallida Bak. (Iridaceae), in rabbits.

Moraea pallida Bak. (yellow tulp) poisoning is the most important plant cardiac glycoside toxicosis in South Africa. The toxic principle, a bufadienolide, is 1α, 2α-epoxyscillirosidine. The aim was to investigate the potential to develop a vaccine against epoxyscillirosidine. Epoxyscillirosidine, proscillaridin and bufalin, were successfully conjugated to hen ovalbumin (OVA), bovine serum albumin (BSA) and keyhole limpet haemocyanin (KLH). There was a low immune response following vaccination of adult male New Zealand White rabbits with epoxyscillirosidine-OVA (n = 3) and OVA (n = 3) using Freund's adjuvant in Trial (T) 1. The immune response improved significantly in T2 following doubling of the dose to 0.8 mg/rabbit and changing the adjuvant to Montanide. In T3, the rabbits (n = 15), allocated into 5 equal groups, vaccinated with proscillaridin-BSA, bufalin-BSA, epoxyscillirosidine-KLH, epoxyscillirosidine-BSA and BSA respectively, using Montanide adjuvant, developed antibodies against the administered immunogens, with epoxyscillirosidine-KLH inducing the highest immune response. Proscillaridin and bufalin antibodies cross-reacted with epoxyscillirosidine in an enzyme linked immunosorbent assay. The conjugation methodology will be adjusted in the future to target optimal conjugation efficiency. Additional vaccination will be conducted in search of neutralizing antibodies against the yellow tulp toxin. The cross-reactivity of proscillaridin and bufalin antibodies with epoxyscillirosidine could be studied in future to explore the potential to prevent yellow tulp poisoning.

Long term stability of a recombinant Plasmodium falciparum AMA1 malaria vaccine adjuvanted with Montanide(®) ISA 720 and stabilized with glycine.

Plasmodium falciparum apical membrane antigen 1 (AMA1) is an asexual blood-stage vaccine candidate against the malaria parasite. AMA1-C1/ISA 720 refers to a mixture of recombinant AMA1 proteins representing the FVO and 3D7 alleles in 1:1 mass ratio, formulated with Montanide(®) ISA 720 as a water-in oil emulsion. In order to develop the AMA1-C1/ISA 720 vaccine for human use, it was important to determine the shelf life of this formulation. Previously it was found 267 mM glycine stabilized the proteins in Montanide(®) ISA 720 formulations for a short period of time at 2-8°C [25]. We now test the long term stability of AMA1-C1 at 10 and 40 µg/mL formulated with Montanide(®) ISA 720 with 50mM glycine as a stabilizer. Stability of AMA1-C1/ISA 720 at different time points following formulation (0, 5, 12 or 18 months) was evaluated by determining the mean particle size (diameter of the mean droplet volume), total protein content by a Modified Lowry assay, identity and integrity using western blot and SDS-PAGE. Our results showed that the mean particle size of these emulsions increased over time, whereas protein content, as determined by an ELISA method using a monoclonal antibody against penta-his, decreased over time. For the 10 µg/mL AMA1-C1/ISA 720 vaccine, the protein content was 6.5±2.2 µg/mL, and for the 40 µg/mL AMA1-C1/ISA 720 vaccine, the protein content was only 8.2±2.3 µg/mL after 18 months of storage at 2-8°C. These results suggest that the integrity of the protein was affected by long-term storage. The results of the present study indicate that the AMA1-C1/ISA 720 emulsion was unstable after 12 months of storage, after which AMA1-C1 proteins were partially degraded.

Modifications increasing the efficacy of recombinant vaccines; marked increase in antibody titers with moderately repetitive variants of a therapeutic allergy vaccine.

Development of vaccines targeting important self-molecules like tumor antigens, IgE, cytokines or other regulatory molecules, brings about challenges that are not met in classical vaccine development. Tolerance inducing mechanisms reduce the levels of therapeutic antibodies in the vaccinated subject, and anti-self antibody titers are frequently more than 50-fold lower than the anti-non-self response to the carrier. In order to overcome this limitation in efficacy we have explored various methods to enhance the immunogenicity of the vaccine antigen. Vaccination with a molecule containing two IgE Cepsilon3 domains and thereby a low level of repetitiveness markedly increased the efficacy. The anti-IgE antibody titers in the animals treated with the dimeric vaccine antigen were 4.5, 5 and 8 times higher than in the animals treated with the monomer, in three independent experiments. In addition, this increase in efficacy was not masked by the use of potent adjuvants. The effect persisted even in the presence of Freunds or Montanide ISA 51, two mineral oil based adjuvants. This in contrast to most Toll-like receptor (TLR) agonists, which appear to enhance the immune response only when administrated together with weak adjuvants. This clearly shows that the introduction of a moderately repetitive structure is enough to substantially increase the efficacy of a therapeutic vaccine.

Peptide based cytotoxic T-cell vaccines; delivery of multiple epitopes, help, memory and problems.

Synthetic CD8+ cytotoxic T-lymphocyte (CTL) peptide epitope based vaccines are being developed against a number of human diseases. Here we describe extensive preclinical testing of peptide epitope vaccines formulated with a protein as a source of CD4 help and Montanide ISA 720, an adjuvant currently in human clinical trials. Such water-in-oil formulations could effectively co-deliver several peptide epitopes and simultaneously induce multiple independent CTL responses. The efficiency of CTL induction by some peptides was, however, dependent on the aqueous buffer conditions, with poor performance correlating with non-covalent peptide oligomerisation. Any of a number of proteins currently used in human vaccines could supply CD4 help and no difference in CTL induction was obtained if the CD4 response was amnestic or a primary. Peptide immunisation was found to induce long term CTL memory and the recall of protective responses did not depend on an amnestic CD4 response. Slow pyroglutamic acid formation and rapid oxidation of methionine residues was observed in water-in-oil formulations, however, the latter had no effect on CTL induction. These data highlight the need to monitor for potential deleterious chemical events and interpeptide interactions, but illustrate that peptide based vaccination can effectively deliver multiple epitopes, in conjunction with any protein, and induce protective memory.

[Multicenter clinical evaluation of red cell concentrates stored up to 6 weeks in MAP, a new additive solution].

The clinical safety and efficacy of transfusion of red cell concentrates stored in MAP solution (MAP-CRC) containing mannitol, adenine, glucose, phosphate and citrate, into 39 anemic patients were evaluated. In 23 patients, infusion of MAP-CRC was alternated with infusion of ordinary CRC as a control. The MAP-CRC and CRC used in this study were stored at 4 degrees C for an average of 38.2 +/- 2.6 days (n = 52) and 18.1 +/- 2.2 days (n = 26), respectively. Red cell recovery was 77.5% for MAP-CRC and 82.5% for CRC, based on calculation of the increase in hemoglobin level one day after transfusion. There were no differences between patients transfused with MAP-CRC and those transfused with CRC in clinical findings or biochemical data. No major side-effects other than pyrexia associated with the underlying infections were seen in patients transfused with MAP-CRC. MAP-CRC stored up to 42 days is apparently as safe and effective as stored CRC. This new additive solution may therefore be useful for the future expansion of the indications for autologous blood transfusion by facilitating the collection and storage of more blood in the liquid state for a longer period, and may also be useful in obtaining more plasma from whole blood as source plasma.