GMMA from genetically-engineered meningococcus as vaccine for Africa

Gram-negative bacteria naturally shed outer membrane vesicles (OMVs). Structural changes in bacterial outer membrane lead to GMMA, Generalized Modules for Membrane Antigens: outer membrane vesicles to use as vaccine. They are safe, cheap to produce, and can give broad protection against pathogens

Copper monitoring in vineyard soils of central Italy subjected to three antifungal treatments, and effects of copper sub-lethal doses on the earthworm Eisenia fetida.

none6The extensive employment of copper-based fungicides has increased copper concentration in vineyard soils. The present study’s objectives were to monitor copper concentration in two vineyard soils during two cropping seasons and study the ecotoxicological effects on the earthworm Eisenia fetida. Total, soluble, and bioavailable copper fractions were measured at the end of two cropping seasons and different depths in two vineyards of central Italy, characterised by three anticryptogamic control methods: copper compounds, chitosan, and combined treatments of them. A laboratory experiment to assess the effects on Eisenia fetida was conducted with soil samples collected in the vineyards with a mean copper concentration of 60 mg/kg and two higher concentrations of 90 and 150 mg/kg. Results showed low levels of total copper concentration in the first 20 cm of soils, regardless of antifungal treatment, highlighting prudent management of the vineyards under study, but the soluble fractions showed a significant increase in all samples during the two cropping seasons. At the dose of 150 mg/kg, earthworms suffer during the first two days, showing weight loss and DNA damage, but they are able to recover until day 28, showing no permanent harm at this copper concentration in soil.openArianna De Bernardi, Enrica Marini, Cristiano Casucci, Luca Tiano, Fabio Marcheggiani, Costantino VischettiDE BERNARDI, Arianna; Marini, Enrica; Casucci, Cristiano; Tiano, Luca; Marcheggiani, Fabio; Vischetti, Costantin

Fluorescent asymmetric bis-ureas for pyrophosphate recognition in pure water

Three fluorescent asymmetric bis-urea receptors (L1–L3) have been synthesised. The binding properties of L1–L3 towards different anions (fluoride, acetate, hydrogencarbonate, dihydrogen phosphate, and hydrogen pyrophosphate HPpi3?) have been studied by means of 1H-NMR, UV-Vis and fluorescence spectroscopy, single crystal X-ray diffraction, and theoretical calculations. In particular, a remarkable affinity for HPpi3? has been observed in the case L1 (DMSO-d6/0.5% H2O) which also acts as a fluorimetric chemosensor for this anion. Interestingly, when L1 is included in cetyltrimethylammonium (CTAB) micelles, hydrogen pyrophosphate recognition can also be achieved in pure water

Combination of RTS,S and Pfs25-IMX313 Induces a Functional Antibody Response Against Malaria Infection and Transmission in Mice

The last two decades saw a dramatic reduction in malaria incidence rates, but this decrease has been stalling recently, indicating control measures are starting to fail. An effective vaccine, particularly one with a marked effect on disease transmission, would undoubtedly be an invaluable tool for efforts to control and eliminate malaria. RTS,S/AS01, the most advanced malaria vaccine to date, targets the parasite before it invades the liver and has the potential to prevent malaria disease as well as transmission by preventing blood stage infection and therefore gametocytogenesis. Unfortunately efficacy in a phase III clinical trial was limited and it is widely believed that a malaria vaccine needed to contain multiple antigens from different life-cycle stages to have a realistic chance of success. A recent study in mice has shown that partially efficacious interventions targeting the pre-erythrocytic and the sexual lifecycle stage synergise in eliminating malaria from a population over multiple generations. Hence, the combination of RTS,S/AS01 with a transmission blocking vaccine (TBV) is highly appealing as a pragmatic and powerful way to increase vaccine efficacy. Here we demonstrate that combining Pfs25-IMX313, one of the TBV candidates currently in clinical development, with RTS,S/AS01 readily induces a functional immune response against both antigens in outbred CD1 mice. Formulation of Pfs25-IMX313 in AS01 significantly increased antibody titres when compared to formulation in Alhydrogel, resulting in improved transmission reducing activity in standard membrane feeding assays (SMFA). Upon co-formulation of Pfs25-IMX313 with RTS,S/AS01, the immunogenicity of both vaccines was maintained, and functional assessment of the induced antibody response by SMFA and inhibition of sporozoite invasion assay (ISI) showed no reduction in biological activity against parasites of both lifecycle stages. Should this findings be translatable to human vaccination this could greatly aid efforts to eliminate and eventually eradicate malaria

Poor CD4+ T Cell Immunogenicity Limits Humoral Immunity to P. falciparum Transmission-Blocking Candidate Pfs25 in Humans.

Plasmodium falciparum transmission-blocking vaccines (TBVs) targeting the Pfs25 antigen have shown promise in mice but the same efficacy has never been achieved in humans. We have previously published pre-clinical data related to a TBV candidate Pfs25-IMX313 encoded in viral vectors which was very promising and hence progressed to human clinical trials. The results from the clinical trial of this vaccine were very modest. Here we unravel why, contrary to mice, this vaccine has failed to induce robust antibody (Ab) titres in humans to elicit transmission-blocking activity. We examined Pfs25-specific B cell and T follicular helper (Tfh) cell responses in mice and humans after vaccination with Pfs25-IMX313 encoded by replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA) delivered in the heterologous prime-boost regimen via intramuscular route. We found that after vaccination, the Pfs25-IMX313 was immunologically suboptimal in humans compared to mice in terms of serum Ab production and antigen-specific B, CD4+ and Tfh cell responses. We identified that the key determinant for the poor anti-Pfs25 Ab formation in humans was the lack of CD4+ T cell recognition of Pfs25-IMX313 derived peptide epitopes. This is supported by correlations established between the ratio of proliferated antigen-specific CD4+/Tfh-like T cells, CXCL13 sera levels, and the corresponding numbers of circulating Pfs25-specific memory B cells, that consequently reflected on antigen-specific IgG sera levels. These correlations can inform the design of next-generation Pfs25-based vaccines for robust and durable blocking of malaria transmission

Contribution of factor H-Binding protein sequence to the cross-reactivity of meningococcal native outer membrane vesicle vaccines with over-expressed fHbp variant group 1

Factor H-binding protein (fHbp) is an important meningococcal vaccine antigen. Native outer membrane vesicles with over-expressed fHbp (NOMV OE fHbp) have been shown to induce antibodies with broader functional activity than recombinant fHbp (rfHbp). Improved understanding of this broad coverage would facilitate rational vaccine design. We performed a pair-wise analysis of 48 surface-exposed amino acids involved in interacting with factor H, among 383 fHbp variant group 1 sequences. We generated isogenic NOMV-producing meningococcal strains from an African serogroup W isolate, each over-expressing one of four fHbp variant group 1 sequences (ID 1, 5, 9, or 74), including those most common among invasive African meningococcal isolates. Mice were immunised with each NOMV, and sera tested for IgG levels against each of the rfHbp ID and for ability to kill a panel of heterologous meningococcal isolates. At the fH-binding site, ID pairs differed by a maximum of 13 (27%) amino acids. ID 9 shared an amino acid sequence common to 83 ID types. The selected ID types differed by up to 6 amino acids, in the fH-binding site. All NOMV and rfHbp induced high IgG levels against each rfHbp. Serum killing from mice immunised with rfHbp was generally less efficient and more restricted compared to NOMV, which induced antibodies that killed most meningococci tested, with decreased stringency for ID type differences. Breadth of killing was mostly due to anti-fHbp antibodies, with some restriction according to ID type sequence differences. Nevertheless, under our experimental conditions, no relationship between antibody cross-reactivity and variation fH-binding site sequence was identified. NOMV over-expressing different fHbp IDs belonging to variant group 1 induce antibodies with fine specificities against fHbp, and ability to kill broadly meningococci expressing heterologous fHbp IDs. The work reinforces that meningococcal NOMV with OE fHbp is a promising vaccine strategy, and provides a basis for rational selection of antigen sequence types for over-expression on NOMV

Safety and Immunogenicity of ChAd63/MVA Pfs25-IMX313 in a Phase I First-in-Human Trial.

BACKGROUND: Transmission blocking vaccines targeting the sexual-stages of the malaria parasite could play a major role to achieve elimination and eradication of malaria. The Plasmodium falciparum Pfs25 protein (Pfs25) is the most clinically advanced candidate sexual-stage antigen. IMX313, a complement inhibitor C4b-binding protein that forms heptamers with the antigen fused to it, improve antibody responses. This is the first time that viral vectors have been used to induce antibodies in humans against an antigen that is expressed only in the mosquito vector. METHODS: Clinical trial looking at safety and immunogenicity of two recombinant viral vectored vaccines encoding Pfs25-IMX313 in healthy malaria-naive adults. Replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA), encoding Pfs25-IMX313, were delivered by the intramuscular route in a heterologous prime-boost regimen using an 8-week interval. Safety data and samples for immunogenicity assays were taken at various time-points. RESULTS: The reactogenicity of the vaccines was similar to that seen in previous trials using the same viral vectors encoding other antigens. The vaccines were immunogenic and induced both antibody and T cell responses against Pfs25, but significant transmission reducing activity (TRA) was not observed in most volunteers by standard membrane feeding assay. CONCLUSION: Both vaccines were well tolerated and demonstrated a favorable safety profile in malaria-naive adults. However, the transmission reducing activity of the antibodies generated were weak, suggesting the need for an alternative vaccine formulation. TRIAL REGISTRATION: Clinicaltrials.gov NCT02532049

Native outer membrane vesicles as vaccine candidates against neisseria meningitidis

An affordable vaccine against all serogroups of Neisseria meningitidis causing reoccurring epidemics in sub-Saharan Africa is needed. Native outer membrane vesicles (NOMV) with over-expressed factor H binding protein (fHbp) represent a promising vaccine approach. We investigated the contribution of fHbp variant 1 amino acidic sequence to the cross-reactivity of the antibody response generated by NOMV OE fHbp. Coupling structure-sequence analysis of fHbp with epidemiological data of meningococcal prevalence in Africa, we selected four fHbp v.1 IDs (1, 5, 9, 74), to generate isogenic mutants of a serogroup W African isolate, over-expressing each of the them. NOMV from the mutants were purified, characterized, and the antibody response generated in mice was investigated, and compared to the corresponding recombinant fHbp. This is the first study indicating that the amino acid sequence of fHbp influences the specificity of the antibody response generated, not only as recombinant protein, but also when over-expressed on NOMV. In mice NOMV OE fHbp induced a fast, long-lasting antibody response, with high IgM and IgG antibody levels 7 days after immunization, and germinal centers induced. LipidA modifications do not impact primary antibody response, while precipitation of the antigen on alum attenuated the early antibody response, but enhance its longevity