Lactobacillli expressing llama VHH fragments neutralise Lactococcus phages

<p>Abstract</p> <p>Background</p> <p>Bacteriophages infecting lactic acid bacteria (LAB) are widely acknowledged as the main cause of milk fermentation failures. In this study, we describe the surface-expression as well as the secretion of two functional llama heavy-chain antibody fragments, one binding to the major capsid protein (MCP) and the other to the receptor-binding proteins (RBP) of the lactococcal bacteriophage p2, by lactobacilli in order to neutralise lactococcal phages.</p> <p>Results</p> <p>The antibody fragment VHH5 that is directed against the RBP, was fused to a c-<it>myc </it>tag and expressed in a secreted form by a <it>Lactobacillus </it>strain. The fragment VHH2 that is binding to the MCP, was fused to an E-tag and anchored on the surface of the lactobacilli. Surface expression of VHH2 was confirmed by flow cytometry using an anti-E-tag antibody. Efficient binding of both the VHH2 and the secreted VHH5 fragment to the phage antigens was shown in ELISA. Scanning electron microscopy showed that lactobacilli expressing VHH2 anchored at their surface were able to bind lactococcal phages. A neutralisation assay also confirmed that the secreted VHH5 and the anchored VHH2 fragments prevented the adsorption of lactococcal phages to their host cells.</p> <p>Conclusion</p> <p>Lactobacilli were able to express functional VHH fragments in both a secreted and a cell surface form and reduced phage infection of lactococcal cells. Lactobacilli expressing llama heavy-chain antibody fragments represent a novel way to limit phage infection.</p

Dual anti-idiotypic purification of a novel, native-format biparatopic anti-MET antibody with improved in vitro and in vivo efficacy

Bispecific antibodies are of great interest due to their ability to simultaneously bind and engage different antigens or epitopes. Nevertheless, it remains a challenge to assemble, produce and/or purify them. Here we present an innovative dual anti-idiotypic purification process, which provides pure bispecific antibodies with native immunoglobulin format. Using this approach, a biparatopic IgG1 antibody targeting two distinct, HGF-competing, non-overlapping epitopes on the extracellular region of the MET receptor, was purified with camelid single-domain antibody fragments that bind specifically to the correct heavy chain/light chain pairings of each arm. The purity and functionality of the anti-MET biparatopic antibody was then confirmed by mass spectrometry and binding experiments, demonstrating its ability to simultaneously target the two epitopes recognized by the parental monoclonal antibodies. The improved MET-inhibitory activity of the biparatopic antibody compared to the parental monoclonal antibodies, was finally corroborated in cell-based assays and more importantly in a tumor xenograft mouse model. In conclusion, this approach is fast and specific, broadly applicable and results in the isolation of a pure, novel and native-format anti-MET biparatopic antibody that shows superior biological activity over the parental monospecific antibodies both in vitro and in vivo

Llama Antibody Fragments Recognizing Various Epitopes of the CD4bs Neutralize a Broad Range of HIV-1 Subtypes A, B and C

Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120Ds2), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B′/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides

Llama-Derived Single Domain Antibodies to Build Multivalent, Superpotent and Broadened Neutralizing Anti-Viral Molecules

For efficient prevention of viral infections and cross protection, simultaneous targeting of multiple viral epitopes is a powerful strategy. Llama heavy chain antibody fragments (VHH) against the trimeric envelope proteins of Respiratory Syncytial Virus (Fusion protein), Rabies virus (Glycoprotein) and H5N1 Influenza (Hemagglutinin 5) were selected from llama derived immune libraries by phage display. Neutralizing VHH recognizing different epitopes in the receptor binding sites on the spikes with affinities in the low nanomolar range were identified for all the three viruses by viral neutralization assays. By fusion of VHH with variable linker lengths, multimeric constructs were made that improved neutralization potencies up to 4,000-fold for RSV, 1,500-fold for Rabies virus and 75-fold for Influenza H5N1. The potencies of the VHH constructs were similar or better than best performing monoclonal antibodies. The cross protection capacity against different viral strains was also improved for all three viruses, both by multivalent (two or three identical VHH) and biparatopic (two different VHH) constructs. By combining a VHH neutralizing RSV subtype A, but not subtype B with a poorly neutralizing VHH with high affinity for subtype B, a biparatopic construct was made with low nanomolar neutralizing potency against both subtypes. Trivalent anti-H5N1 VHH neutralized both Influenza H5N1 clade1 and 2 in a pseudotype assay and was very potent in neutralizing the NIBRG-14 Influenza H5N1 strain with IC50 of 9 picomolar. Bivalent and biparatopic constructs against Rabies virus cross neutralized both 10 different Genotype 1 strains and Genotype 5. The results show that multimerization of VHH fragments targeting multiple epitopes on a viral trimeric spike protein is a powerful tool for anti-viral therapy to achieve "best-in-class" and broader neutralization capacity

Treatment of lakes and storage reservoirs with very low dosages of selenium to reduce methyl mercury in fish

The trials of the addition of selenium to three lakes indicate a rapid bioackumulation of selenium. The selenium concentrations in zooplankton increased, and these constitute the first and most important level in the aquatic ecosystem. The increase of selenium in zooplankton resulted in a reduction in the concentration of methyl mercury. The bio-magnification in the food chain led to increases in selenium concentrations and reductions in methyl mercury. This in turn led to reduced concentrations of mercury in crayfish and methyl mercury in younger fish. After a certain delay, the bio-magnification of selenium continued in the higher trophic levels. This resulted in increased selenium concentrations and reduced methyl mercury concentrations in older perch and pike several years after the zooplankton, young roach and young perch. When the selenium treatments stopped the concentrations of selenium in the water, aquatic moss, bentic invertebrates and the youngest age groups of roach and perch fell immediately. The reductions in selenium in these parts of the ecosystem are related to the water retention period of the lake or reservoir.The trials of the addition of selenium to three lakes indicate a rapid bioackumulation of selenium. The selenium concentrations in zooplankton increased, and these constitute the first and most important level in the aquatic ecosystem. The increase of selenium in zooplankton resulted in a reduction in the concentration of methyl mercury. The bio-magnification in the food chain led to increases in selenium concentrations and reductions in methyl mercury. This in turn led to reduced concentrations of mercury in crayfish and methyl mercury in younger fish. After a certain delay, the bio-magnification of selenium continued in the higher trophic levels. This resulted in increased selenium concentrations and reduced methyl mercury concentrations in older perch and pike several years after the zooplankton, young roach and young perch. When the selenium treatments stopped the concentrations of selenium in the water, aquatic moss, bentic invertebrates and the youngest age groups of roach and perch fell immediately. The reductions in selenium in these parts of the ecosystem are related to the water retention period of the lake or reservoir

Behandling med låg-låg dos av selen i sjöar och regleringsmagasin för att minska halten metylkvicksilver i fisk

De genomförda försöken med tillförsel av selen i tre sjöar visar på en snabb bioackumulation av selen. Ökningen av selen sker redan i djurplankton som är det första och viktigaste nivån i det akvatiska ekosystemet. Ökningen av selen i djurplankton medför att halten av metylkvicksilver minskar. Via biomagnifiering i näringskedjan ökar selenhalterna och metylkvicksilver minskar. I sin tur medför detta minskad halt av kvicksilver i flodkräfta och metylkvicksilver i yngre fisk. Med en tidsfödröjning fortsätter biomagnifieringen av selen allt högre upp i trofiniovåerna. Detta medför att äldre abborre och gäddor får ökade halter av selen och minskade halter av metyylkvicksilver flera år efter djurplankton och unga mörtar och unga abborrar. Då tillförseln av selen upphör minskar halterna omgående i vatten, bäckmossa, lägre bottelevande djur, djurplankton och de yngsta årsklasserna av mört och abborre. Haltminskningen i dessa delar av ekosystemet följer sjöns eller magasinets omsättningstid av vatten.De genomförda försöken med tillförsel av selen i tre sjöar visar på en snabb bioackumulation av selen. Ökningen av selen sker redan i djurplankton som är det första och viktigaste nivån i det akvatiska ekosystemet. Ökningen av selen i djurplankton medför att halten av metylkvicksilver minskar. Via biomagnifiering i näringskedjan ökar selenhalterna och metylkvicksilver minskar. I sin tur medför detta minskad halt av kvicksilver i flodkräfta och metylkvicksilver i yngre fisk. Med en tidsfödröjning fortsätter biomagnifieringen av selen allt högre upp i trofiniovåerna. Detta medför att äldre abborre och gäddor får ökade halter av selen och minskade halter av metyylkvicksilver flera år efter djurplankton och unga mörtar och unga abborrar. Då tillförseln av selen upphör minskar halterna omgående i vatten, bäckmossa, lägre bottelevande djur, djurplankton och de yngsta årsklasserna av mört och abborre. Haltminskningen i dessa delar av ekosystemet följer sjöns eller magasinets omsättningstid av vatten

Treatment of lakes and storage reservoirs with very low dosages of selenium to reduce methyl mercury in fish

The trials of the addition of selenium to three lakes indicate a rapid bioackumulation of selenium. The selenium concentrations in zooplankton increased, and these constitute the first and most important level in the aquatic ecosystem. The increase of selenium in zooplankton resulted in a reduction in the concentration of methyl mercury. The bio-magnification in the food chain led to increases in selenium concentrations and reductions in methyl mercury. This in turn led to reduced concentrations of mercury in crayfish and methyl mercury in younger fish. After a certain delay, the bio-magnification of selenium continued in the higher trophic levels. This resulted in increased selenium concentrations and reduced methyl mercury concentrations in older perch and pike several years after the zooplankton, young roach and young perch. When the selenium treatments stopped the concentrations of selenium in the water, aquatic moss, bentic invertebrates and the youngest age groups of roach and perch fell immediately. The reductions in selenium in these parts of the ecosystem are related to the water retention period of the lake or reservoir.The trials of the addition of selenium to three lakes indicate a rapid bioackumulation of selenium. The selenium concentrations in zooplankton increased, and these constitute the first and most important level in the aquatic ecosystem. The increase of selenium in zooplankton resulted in a reduction in the concentration of methyl mercury. The bio-magnification in the food chain led to increases in selenium concentrations and reductions in methyl mercury. This in turn led to reduced concentrations of mercury in crayfish and methyl mercury in younger fish. After a certain delay, the bio-magnification of selenium continued in the higher trophic levels. This resulted in increased selenium concentrations and reduced methyl mercury concentrations in older perch and pike several years after the zooplankton, young roach and young perch. When the selenium treatments stopped the concentrations of selenium in the water, aquatic moss, bentic invertebrates and the youngest age groups of roach and perch fell immediately. The reductions in selenium in these parts of the ecosystem are related to the water retention period of the lake or reservoir