Preclinical immunogenicity and safety of the cGMP-grade placental malaria vaccine PRIMVAC.

BACKGROUND: VAR2CSA is the lead antigen for developing a vaccine that would protect pregnant women against placental malaria. A multi-system feasibility study has identified E. coli as a suitable bacterial expression platform allowing the production of recombinant VAR2CSA-DBL1x-2x (PRIMVAC) to envisage a prompt transition to current Good Manufacturing Practice (cGMP) vaccine production. METHODS: Extensive process developments were undertaken to produce cGMP grade PRIMVAC to permit early phase clinical trials. PRIMVAC stability upon storage was assessed over up to 3 years. A broad toxicology investigation was carried out in rats allowing meanwhile the analysis of PRIMVAC immunogenicity. FINDINGS: We describe the successful cGMP production of 4. 65 g of PRIMVAC. PRIMVAC drug product was stable and potent for up to 3 years upon storage at -20 °C and showed an absence of toxicity in rats. PRIMVAC adjuvanted with Alhydrogel® or GLA-SE was able to generate antibodies able to recognize VAR2CSA expressed at the surface of erythrocytes infected with different strains. These antibodies also inhibit the interaction of the homologous NF54-CSA strain and to a lower extend of heterologous strains to CSA. INTERPRETATION: This work paved the way for the clinical development of an easily scalable low cost effective vaccine that could protect against placental malaria and prevent an estimated 10,000 maternal and 200,000 infant deaths annually. FUND: This work was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF), Germany through Kreditanstalt für Wiederaufbau (KfW) (Reference No: 202060457) and through funding from Irish Aid, Department of Foreign Affairs and Trade, Ireland.

Quantification of Demodex folliculorum by PCR in rosacea and its relationship to skin innate immune activation.

The aim of this study is to quantify D. folliculorum colonisation in rosacea subtypes and age-matched controls and to determine the relationship between D. folliculorum load, rosacea subtype and skin innate immune system activation markers. We set up a multicentre, cross-sectional, prospective study in which 98 adults were included: 50 with facial rosacea, including 18 with erythematotelangiectatic rosacea (ETR), and 32 with papulopustular rosacea (PPR) and 48 age- and sex-matched healthy volunteers. Non-invasive facial samples were taken to quantify D. folliculorum infestation by quantitative PCR and evaluate inflammatory and immune markers. Analysis of the skin samples show that D. folliculorum was detected more frequently in rosacea patients than age-matched controls (96% vs 74%, P < 0.01). D. folliculorum density was 5.7 times higher in rosacea patients than in healthy volunteers. Skin sample analysis showed a higher expression of genes encoding pro-inflammatory cytokines (Il-8, Il-1b, TNF-a) and inflammasome-related genes (NALP-3 and CASP-1) in rosacea, especially PPR. Overexpression of LL-37 and VEGF, as well as CD45RO, MPO and CD163, was observed, indicating broad immune system activation in patients with rosacea. In conclusion, D. folliculorum density is highly increased in patients with rosacea, irrespective of rosacea subtype. There appears to be an inverse relationship between D. folliculorum density and inflammation markers in the skin of rosacea patients, with clear differences between rosacea subtypes.

PpiA, a surface PPIase of the cyclophilin family in Lactococcus lactis.

BACKGROUND: Protein folding in the envelope is a crucial limiting step of protein export and secretion. In order to better understand this process in Lactococcus lactis, a lactic acid bacterium, genes encoding putative exported folding factors like Peptidyl Prolyl Isomerases (PPIases) were searched for in lactococcal genomes. RESULTS: In L. lactis, a new putative membrane PPIase of the cyclophilin subfamily, PpiA, was identified and characterized. ppiA gene was found to be constitutively expressed under normal and stress (heat shock, H(2)O(2)) conditions. Under normal conditions, PpiA protein was synthesized and released from intact cells by an exogenously added protease, showing that it was exposed at the cell surface. No obvious phenotype could be associated to a ppiA mutant strain under several laboratory conditions including stress conditions, except a very low sensitivity to H(2)O(2). Induction of a ppiA copy provided in trans had no effect i) on the thermosensitivity of an mutant strain deficient for the lactococcal surface protease HtrA and ii) on the secretion and stability on four exported proteins (a highly degraded hybrid protein and three heterologous secreted proteins) in an otherwise wild-type strain background. However, a recombinant soluble form of PpiA that had been produced and secreted in L. lactis and purified from a culture supernatant displayed both PPIase and chaperone activities. CONCLUSIONS: Although L. lactis PpiA, a protein produced and exposed at the cell surface under normal conditions, displayed a very moderate role in vivo, it was found, as a recombinant soluble form, to be endowed with folding activities in vitro.

Production and purification of staphylococcal nuclease in Lactococcus lactis using a new expression-secretion system and a pH-regulated mini-reactor.

BACKGROUND: Staphylococcal (or micrococcal) nuclease or thermonuclease (SNase or Nuc) is a naturally-secreted nucleic acid degrading enzyme that participates in Staphylococcus aureus spread in the infected host. Purified Nuc protein can be used as an exogenous reagent to clear cellular extracts and improve protein purification. Here, a recombinant form of Nuc was produced and secreted in a Gram-positive host, Lactococcus lactis, and purified from the culture medium. RESULTS: The gene segment corresponding to the S. aureus nuclease without its signal peptide was cloned in an expression-secretion vector. It was then fused to a lactococcal sequence encoding a signal peptide, and expressed under the control of a lactococcal promoter that is inducible by zinc starvation. An L. lactis subsp cremoris model strain (MG1363) transformed with the resulting plasmid was grown in either of two media (GM17v and CDM) that are free of animal compounds, allowing GMP (Good Manufacturing Practice) production. Induction conditions (concentration of the metal chelator EDTA and timing of addition) in small-scale pH-regulated fermentors were optimized using LacMF (Lactis Multi-Fermentor), a home-made parallel fermentation control system able to monitor 12 reactors simultaneously. Large amounts of recombinant Nuc (rNuc) were produced and secreted in both media, and rNuc was purified from GM17v medium in a single-step procedure. CONCLUSIONS: In L. lactis, rNuc production and secretion were optimal after induction by 0.5 mM EDTA in small scale (200 mL) GM17v exponential phase cultures (at an OD(600) of 2), leading to a maximal protein yield of 210 mg per L of culture medium. Purified rNuc was highly active, displaying a specific activity of 2000 U/mg.

Videokeratographic anomalies in familial keratoconus.

OBJECTIVE: To analyze videokeratography of relatives of established familial keratoconus (FK) patients to detect low-expressivity keratoconus and improve the diagnosis criteria of forme fruste keratoconus. DESIGN: Multicenter case-control study. PARTICIPANTS: Twenty-three families with 55 clinical keratoconus patients, 89 first-degree relatives, 43 other relatives, and a control group of 130 subjects. METHODS: Videokeratography was performed on both eyes of patients after clinical examination, and corneal maps were analyzed. Statistical comparisons were conducted between first-degree and other relatives and a control population. MAIN OUTCOME MEASURES: Qualitative (using a 0.5-diopter [D] increment scale) and quantitative analyses of videokeratographs. RESULTS: Two corneal patterns were overrepresented in the relatives of FK patients: the J and inverted-J form patterns. Results of the quantitative analysis of these suspect patterns showed that the inferior - superior values (reflecting the inferior - superior dioptric asymmetry) were close to 0.8 D and the Srax (relative skewing of the steepest radial axes) was superior to 21 degrees. CONCLUSION: Our study using topography in clearly established genetic keratoconus families allowed us to detect suspect topographical patterns and brings new data to the difficult task of diagnosing forme fruste keratoconus.