ABSTRACT Influenza virus still represents a serious threat for human health, causing pandemics an... more ABSTRACT Influenza virus still represents a serious threat for human health, causing pandemics and annual epidemics. From 1918, four influenza A pandemics occurred, caused by H1N1, H2N2, H3N2 and vH1N1 subtypes. Moreover, the recurrent concerns deriving from the possible adaptation to humans of influenza viral isolates circulating in animal reservoirs, the potential arising of resistance to the currently available anti-viral drugs and the lack of an effective prevention make influenza virus a relevant menace for the world population. In this scenario, the identification of human monoclonal antibodies endowed with strong neutralizing activity and directed against viral isolates belonging to different influenza subtypes can raise hope for the development of monoclonal antibody-based immunotherapy. Even more interestingly, they can play a pivotal role in the development of new vaccines. Here, we describe two human mAbs directed against influenza haemagglutinin (HA), named PN-SIA28 and PN-SIA49, featuring such characteristics. They can be effectively considered as suitable candidates for antibody-based immunotherapy due to their low IC50 (half maximal (50%) inhibitory concentration) and to the protection of animal models against lethal influenza virus challenges conferred by PN-SIA49. At the same time, the fine characterization of the conserved structural motifs on the HA, recognized by these mAbs, can be the key to success in the development of new epitope-based vaccines.
Kato-Katz is a widely used method for the diagnosis of soil-transmitted helminth infection. Fecal... more Kato-Katz is a widely used method for the diagnosis of soil-transmitted helminth infection. Fecal samples cannot be preserved, and hence, should be processed on the day of collection and examined under a microscope within 60 min of slide preparation. Mini-FLOTAC is a technique that allows examining fixed fecal samples. We assessed the performance of Mini-FLOTAC using formalin-fixed stool samples compared to Kato-Katz and determined the dynamics of prevalence and intensity estimates of soil-transmitted helminth infection over a 31-day time period. The study was carried out in late 2013 on Pemba Island, Tanzania. Forty-one children were enrolled and stool samples were subjected on the day of collection to a single Kato-Katz thick smear and Mini-FLOTAC examination; 12 aliquots of stool were fixed in 5% formalin and subsequently examined by Mini-FLOTAC up to 31 days after collection. The combined results from Kato-Katz and Mini-FLOTAC revealed that 100% of children were positive for Tri...
The pandemic caused by the new H1N1 swine-origin influenza virus (S-OIV) strain is a worldwide he... more The pandemic caused by the new H1N1 swine-origin influenza virus (S-OIV) strain is a worldwide health emergency and alternative therapeutic and prophylactic options are greatly needed. Two human monoclonal antibody Fab fragments (HMab) neutralizing the novel H1N1 influenza strain at very low concentrations were cloned from a patient who had a broad-range anti-H1N1 serum neutralizing activity. The two HMabs neutralized S-OIV with an IC50 of 2.8 and 4 microg/mL. The genes coding for the neutralizing HMabs could be used for generating full human monoclonal IgGs that can be safely administered with the potentially of representing a novel drug to be used in the prophylaxis and the treatment of this human infection. This is the first report of molecular cloning of human monoclonal antibodies against the new pandemic swine-origin influenza virus.
The preparation of random combinatorial libraries exposed on the surface of phage provides a rout... more The preparation of random combinatorial libraries exposed on the surface of phage provides a route for the selection of diverse high affinity human monoclonal antibodies. However, in particular settings, the isolation of genes coding for a rare antibody can be elusive because some epitopes are predominant and because, in the case of impure antigens, the protein or any compound of interest can be present in relatively minimal amount. In this paper, we describe the successful utilization of a new strategy of "preadsorption" panning that allowed us to clone a rare human monoclonal antibody fragment and to access a different antibody repertoire. The procedure is easy, fast, inexpensive, can be used together with other panning techniques and can be particularly useful in cloning antibodies against rare or unknown determinants.
It is well known that immunoglobulins with no identifiable immunogenic origin, called natural ant... more It is well known that immunoglobulins with no identifiable immunogenic origin, called natural antibodies, are present in the sera of healthy individuals and their role as a defence against important pathogens has been proposed. Unfortunately, the studies are hampered by the fact that these immunoglobulins seem to have low affinity and to be polyreactive, and are commonly available in polyclonal preparations. Lately, new technologies for the production of monoclonal antibodies became available, and in particular the cloning of genes coding for antibody fragments in combinatorial phage display vectors provided a handy tool for the selection of human monoclonal antibodies. In this work, we describe the successful development of a technology for the molecular cloning of combinatorial phage display libraries containing genes coding exclusively for antibody fragment of the IgM or IgD phenotype. These libraries can be useful for molecular cloning of monoclonal antibodies of the IgM and IgD...
Phagemid pComb3 is a widely used vector for molecular cloning of the antibody repertoire and for ... more Phagemid pComb3 is a widely used vector for molecular cloning of the antibody repertoire and for production of phage display libraries. However, in practical use, the utilization of this vector has some drawbacks. In this work we describe the construction of pComb3/TIG, an improved, easily manipulated vector for the cloning and display of antibody fragment libraries on the surface of filamentous phage. The two small "stuffer" fragments at the cloning sites were replaced with long DNA fragments, for easier differentiation of the correctly cut forms of the vector. Moreover, in pComb3/TIG the fragment at the heavy-chain-fragment cloning site contains an acid phosphatase-encoding gene. This feature allows the easy distinction of the Escherichia coli cells containing the unmodified form of the phagemid instead of the heavy-chain fragment coding cDNA in a simple plate histochemical assay.
The determination and quantitation of peripheral blood leukocytes (PBLs) expressing human cytomeg... more The determination and quantitation of peripheral blood leukocytes (PBLs) expressing human cytomegalo- virus (HCMV) antigens is widely employed in clinical virology for rapid diagnosis of HCMV-related infections. We describe how CMV antigenemia may be accurately detected by means of human recombinant monoclonal Fab fragments rescued from a combinatorial phage display library prepared from an HCMV-infected donor. Fourteen recombinant Fabs were
Proceedings of the National Academy of Sciences, 1994
Herpes simplex viruses 1 and 2 (HSV-1 and -2) are associated with a number of conditions of varyi... more Herpes simplex viruses 1 and 2 (HSV-1 and -2) are associated with a number of conditions of varying severity, which are only partially responsive to current therapies. Human antibodies to the viruses offer a potential alternative. We describe here the generation of panels of human monoclonal Fab fragments to HSV-1 and -2 by panning a phage display combinatorial antibody library against whole lysates from the two viruses. Each lysate selected a largely distinct set of Fabs, although all of the Fabs were cross-reactive with both viruses. In a plaque-reduction assay, one Fab neutralized HSV-1 at 0.25 microgram/ml (50% reduction) and HSV-2 at 0.05 microgram/ml. This Fab also inhibited plaque formation when applied to virus-infected monolayers, completely abolishing HSV-2 plaque development at 25 micrograms/ml 72 hr postinfection, indicating the ability of the Fab to prevent cell-to-cell spread of virus. The Fab was shown to recognize viral glycoprotein D and to neutralize virus primarily by a postattachment mechanism. Recombinant Fabs may be useful for topical administration, although whole antibody will probably be required for systemic use.
ABSTRACT Influenza virus still represents a serious threat for human health, causing pandemics an... more ABSTRACT Influenza virus still represents a serious threat for human health, causing pandemics and annual epidemics. From 1918, four influenza A pandemics occurred, caused by H1N1, H2N2, H3N2 and vH1N1 subtypes. Moreover, the recurrent concerns deriving from the possible adaptation to humans of influenza viral isolates circulating in animal reservoirs, the potential arising of resistance to the currently available anti-viral drugs and the lack of an effective prevention make influenza virus a relevant menace for the world population. In this scenario, the identification of human monoclonal antibodies endowed with strong neutralizing activity and directed against viral isolates belonging to different influenza subtypes can raise hope for the development of monoclonal antibody-based immunotherapy. Even more interestingly, they can play a pivotal role in the development of new vaccines. Here, we describe two human mAbs directed against influenza haemagglutinin (HA), named PN-SIA28 and PN-SIA49, featuring such characteristics. They can be effectively considered as suitable candidates for antibody-based immunotherapy due to their low IC50 (half maximal (50%) inhibitory concentration) and to the protection of animal models against lethal influenza virus challenges conferred by PN-SIA49. At the same time, the fine characterization of the conserved structural motifs on the HA, recognized by these mAbs, can be the key to success in the development of new epitope-based vaccines.
Kato-Katz is a widely used method for the diagnosis of soil-transmitted helminth infection. Fecal... more Kato-Katz is a widely used method for the diagnosis of soil-transmitted helminth infection. Fecal samples cannot be preserved, and hence, should be processed on the day of collection and examined under a microscope within 60 min of slide preparation. Mini-FLOTAC is a technique that allows examining fixed fecal samples. We assessed the performance of Mini-FLOTAC using formalin-fixed stool samples compared to Kato-Katz and determined the dynamics of prevalence and intensity estimates of soil-transmitted helminth infection over a 31-day time period. The study was carried out in late 2013 on Pemba Island, Tanzania. Forty-one children were enrolled and stool samples were subjected on the day of collection to a single Kato-Katz thick smear and Mini-FLOTAC examination; 12 aliquots of stool were fixed in 5% formalin and subsequently examined by Mini-FLOTAC up to 31 days after collection. The combined results from Kato-Katz and Mini-FLOTAC revealed that 100% of children were positive for Tri...
The pandemic caused by the new H1N1 swine-origin influenza virus (S-OIV) strain is a worldwide he... more The pandemic caused by the new H1N1 swine-origin influenza virus (S-OIV) strain is a worldwide health emergency and alternative therapeutic and prophylactic options are greatly needed. Two human monoclonal antibody Fab fragments (HMab) neutralizing the novel H1N1 influenza strain at very low concentrations were cloned from a patient who had a broad-range anti-H1N1 serum neutralizing activity. The two HMabs neutralized S-OIV with an IC50 of 2.8 and 4 microg/mL. The genes coding for the neutralizing HMabs could be used for generating full human monoclonal IgGs that can be safely administered with the potentially of representing a novel drug to be used in the prophylaxis and the treatment of this human infection. This is the first report of molecular cloning of human monoclonal antibodies against the new pandemic swine-origin influenza virus.
The preparation of random combinatorial libraries exposed on the surface of phage provides a rout... more The preparation of random combinatorial libraries exposed on the surface of phage provides a route for the selection of diverse high affinity human monoclonal antibodies. However, in particular settings, the isolation of genes coding for a rare antibody can be elusive because some epitopes are predominant and because, in the case of impure antigens, the protein or any compound of interest can be present in relatively minimal amount. In this paper, we describe the successful utilization of a new strategy of "preadsorption" panning that allowed us to clone a rare human monoclonal antibody fragment and to access a different antibody repertoire. The procedure is easy, fast, inexpensive, can be used together with other panning techniques and can be particularly useful in cloning antibodies against rare or unknown determinants.
It is well known that immunoglobulins with no identifiable immunogenic origin, called natural ant... more It is well known that immunoglobulins with no identifiable immunogenic origin, called natural antibodies, are present in the sera of healthy individuals and their role as a defence against important pathogens has been proposed. Unfortunately, the studies are hampered by the fact that these immunoglobulins seem to have low affinity and to be polyreactive, and are commonly available in polyclonal preparations. Lately, new technologies for the production of monoclonal antibodies became available, and in particular the cloning of genes coding for antibody fragments in combinatorial phage display vectors provided a handy tool for the selection of human monoclonal antibodies. In this work, we describe the successful development of a technology for the molecular cloning of combinatorial phage display libraries containing genes coding exclusively for antibody fragment of the IgM or IgD phenotype. These libraries can be useful for molecular cloning of monoclonal antibodies of the IgM and IgD...
Phagemid pComb3 is a widely used vector for molecular cloning of the antibody repertoire and for ... more Phagemid pComb3 is a widely used vector for molecular cloning of the antibody repertoire and for production of phage display libraries. However, in practical use, the utilization of this vector has some drawbacks. In this work we describe the construction of pComb3/TIG, an improved, easily manipulated vector for the cloning and display of antibody fragment libraries on the surface of filamentous phage. The two small "stuffer" fragments at the cloning sites were replaced with long DNA fragments, for easier differentiation of the correctly cut forms of the vector. Moreover, in pComb3/TIG the fragment at the heavy-chain-fragment cloning site contains an acid phosphatase-encoding gene. This feature allows the easy distinction of the Escherichia coli cells containing the unmodified form of the phagemid instead of the heavy-chain fragment coding cDNA in a simple plate histochemical assay.
The determination and quantitation of peripheral blood leukocytes (PBLs) expressing human cytomeg... more The determination and quantitation of peripheral blood leukocytes (PBLs) expressing human cytomegalo- virus (HCMV) antigens is widely employed in clinical virology for rapid diagnosis of HCMV-related infections. We describe how CMV antigenemia may be accurately detected by means of human recombinant monoclonal Fab fragments rescued from a combinatorial phage display library prepared from an HCMV-infected donor. Fourteen recombinant Fabs were
Proceedings of the National Academy of Sciences, 1994
Herpes simplex viruses 1 and 2 (HSV-1 and -2) are associated with a number of conditions of varyi... more Herpes simplex viruses 1 and 2 (HSV-1 and -2) are associated with a number of conditions of varying severity, which are only partially responsive to current therapies. Human antibodies to the viruses offer a potential alternative. We describe here the generation of panels of human monoclonal Fab fragments to HSV-1 and -2 by panning a phage display combinatorial antibody library against whole lysates from the two viruses. Each lysate selected a largely distinct set of Fabs, although all of the Fabs were cross-reactive with both viruses. In a plaque-reduction assay, one Fab neutralized HSV-1 at 0.25 microgram/ml (50% reduction) and HSV-2 at 0.05 microgram/ml. This Fab also inhibited plaque formation when applied to virus-infected monolayers, completely abolishing HSV-2 plaque development at 25 micrograms/ml 72 hr postinfection, indicating the ability of the Fab to prevent cell-to-cell spread of virus. The Fab was shown to recognize viral glycoprotein D and to neutralize virus primarily by a postattachment mechanism. Recombinant Fabs may be useful for topical administration, although whole antibody will probably be required for systemic use.
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