Molecular profile of a human monoclonal antibody Fab fragment specific for Epstein-Barr virus gp350/220 antigen.

Experimental evidence indicates Epstein Barr virus (EBV) envelope glycoprotein gp350/220 elicits a potent virus neutralizing response in the infected human host that may play an important role in restricting viral pathogenesis. In this study, we report the molecular cloning in combinatorial phage display vectors, of the IgG1 repertoire of an individual naturally infected with EBV, and describe the recovery and characterization of a monoclonal antibody recognizing gp350/220. A detailed understanding of the human antibody response in EBV infection will identify antibodies of potential use in anti-viral prophylaxis and will advance the production of more effective vaccine candidates.

Cloning the antibody response in humans with inflammatory CNS disease: isolation of measles virus-specific antibodies from phage display libraries of a subacute sclerosing panencephalitis brain.

We have developed a strategy to identify the disease-relevant antigens in a chronic inflammatory CNS disease exhibiting intrathecally expressed oligoclonal IgG. Using subacute sclerosing panencephalitis (SSPE), a chronic inflammatory measles virus infection of the brain as a model system, we constructed a phage display antibody Fab library from the amplified products of IgG expressed in the brain. Selection of the library against measles virus-infected cell lysates yielded four distinct Fabs which, by ELISA and by immunostaining, reacted specifically with measles virus-infected cells. Three Fabs immunoprecipitated a 72 kDa protein from infected cell cultures corresponding to the measles virus phosphoprotein. The fourth Fab immunoprecipitated and recognized by immunoblotting a 60 kDa protein corresponding to the measles virus nucleoprotein. The results demonstrate that functional antibodies from an inflammatory CNS disease can be expressed in bacteria and used to identify disease-relevant antigens. This approach could be applied to chronic inflammatory CNS diseases of unknown cause such as multiple sclerosis.

Human antibody responses to mature and immature forms of viral envelope in respiratory syncytial virus infection: significance for subunit vaccines.

A number of antibodies generated during human respiratory syncytial virus (RSV) infection have been cloned by the phage library approach. Antibodies reactive with an immunodominant epitope on the F glycoprotein of this virus have a high affinity for affinity-purified F antigen. These antibodies, however, have a much lower affinity for mature F glycoprotein on the surface of infected cells and are nonneutralizing. In contrast, a potent neutralizing antibody has a high affinity for mature F protein but a much lower affinity for purified F protein or F protein in viral lysates. The data indicate that at least two F protein immunogens are produced during natural RSV infection: immature F, found in viral lysates, and mature F, found on infected cells or virions. Binding studies with polyclonal human immunoglobulin G suggest that the antibody responses to the two immunogens are of similar magnitudes. Competitive binding studies suggest that overlap between the responses is relatively limited. A mature envelope with an antigenic configuration different from that of the immature envelope has an evolutionary advantage in that the infecting virus is less subject to neutralization by the humoral response to the immature envelope that inevitably arises following lysis of infected cells. Subunit vaccines may be at a disadvantage because they most often resemble immature envelope molecules and ignore this aspect of viral evasion.

Mapping the prion protein using recombinant antibodies.

The fundamental event in prion disease is thought to be the posttranslational conversion of the cellular prion protein (PrPC) into a pathogenic isoform (PrPSc). The occurrence of PrPC on the cell surface and PrPSc in amyloid plaques in situ or in aggregates following purification complicates the study of the molecular events that underlie the disease process. Monoclonal antibodies are highly sensitive probes of protein conformation which can be used under these conditions. Here, we report the rescue of a diverse panel of 19 PrP-specific recombinant monoclonal antibodies from phage display libraries prepared from PrP deficient (Prnp0/0) mice immunized with infectious prions either in the form of rods or PrP 27-30 dispersed into liposomes. The antibodies recognize a number of distinct linear and discontinuous epitopes that are presented to a varying degree on different PrP preparations. The epitope reactivity of the recombinant PrP(90-231) molecule was almost indistinguishable from that of PrPC on the cell surface, validating the importance of detailed structural studies on the recombinant molecule. Only one epitope region at the C terminus of PrP was well presented on both PrPC and PrPSc, while epitopes associated with most of the antibodies in the panel were present on PrPC but absent from PrPSc.

A conservative surgical approach of osteochondroma affecting the mandibular condyle.

An osteochondroma of the condyle in a 49-year-old venezuelan female patients is reported. Clinical, radiological and histopathological features of the tumor are described. A wide local surgical excision permitted the patient to regain normal function.

A conformational transition at the N terminus of the prion protein features in formation of the scrapie isoform.

The scrapie prion protein (PrPSc) is formed from the cellular isoform (PrPC) by a post-translational process that involves a profound conformational change. Linear epitopes for recombinant antibody Fab fragments (Fabs) on PrPC and on the protease-resistant core of PrPSc, designated PrP 27-30, were identified using ELISA and immunoprecipitation. An epitope region at the C terminus was accessible in both PrPC and PrP 27-30; in contrast, epitopes towards the N-terminal region (residues 90 to 120) were accessible in PrPC but largely cryptic in PrP 27-30. Denaturation of PrP 27-30 exposed the epitopes of the N-terminal domain. We argue from our findings that the major conformational change underlying PrPSc formation occurs within the N-terminal segment of PrP 27-30.

Use of recombinant human antibody fragments for detection of cytomegalovirus antigenemia.

The determination and quantitation of peripheral blood leukocytes (PBLs) expressing human cytomegalovirus (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 tested against HCMV-positive PBLs from a patient with ongoing HCMV infection. Three clones were found to react specifically with the nuclei of these cells. These three recombinant Fabs were subsequently tested, individually and pooled together, against 60 PBL samples taken from immunosuppressed patients. The reactivity observed was comparable to that obtained with mouse monoclonal antibodies commercially available for this purpose. The three recombinant Fabs were shown to react specifically with the 65-kDa viral tegument phosphoprotein encoded by UL83 (pUL83), which is the most abundant viral antigen in HCMV-infected PBLs.

Topically applied human recombinant monoclonal IgG1 antibody and its Fab and F(ab')2 fragments protect mice from vaginal transmission of HSV-2.

A recombinant human anti-herpes simplex virus monoclonal IgG1, antibody and the corresponding Fab and F(ab')2 fragments were tested for efficacy in preventing vaginal transmission of HSV-2 infection in a well-established mouse model for genital herpes. IgG1, Fab, and F(ab')2 were approximately equally protective; vaginal delivery of 1-5 ng provided approximately 50% protection, and vaginal delivery of 400 ng completely protected mice from genital herpes infection (P < 0.001). These results suggest that topical applications of human monoclonal antibodies may be useful in developing new methods for preventing sexually transmitted disease.

Circumventing tolerance to generate autologous monoclonal antibodies to the prion protein.

Prion diseases are disorders of protein conformation and do not provoke an immune response. Raising antibodies to the prion protein (PrP) has been difficult due to conservation of the PrP sequence and to inhibitory activity of alpha-PrP antibodies toward lymphocytes. To circumvent these problems, we immunized mice in which the PrP gene was ablated (Prnp 0/0) and retrieved specific monoclonal antibodies (mAbs) through phage display libraries. This approach yielded alpha-PrP mAbs that recognize mouse PrP. Studies with these mAbs suggest that cellular PrP adopts an unusually open structure consistent with the conformational plasticity of this protein.

[Mosquito fauna (Diptera:Culicidae) from Falcon State, Venezuela. I. New records and current checklist]. / Fauna de mosquitos (Diptera:Culicidae) del Estado Falcon, Venezuela. I. Nuevos registros y listado general de especies.

A total of 16 new species records of Culicidae from Falcon State was collected at the "Juan Crisostomo Falcon National Park" (Sierra de San Luis), Natural Monument "Cerro Santa Ana", Coro, and La Vela. Species of Sabethini, Culicini and Toxorhynchitini Tribes were found in natural breeding sites (Phytotelmata), with special occurrence in plants belonging to Tillandsia, Vriesea, Guzmania, Aechmea (Bromelianceae), Heliconia (Heliconiaceae), Calathea (Marantaceae) and Colocasia (Araceae). Aedini and Mansonini were collected only as adults. A specie of Culex (Carrollia) was collected from an artificial container. The Culicidae species belong to 6 genera out of the 23 genera reported from Venezuela (Culex, Wyeomyia, Johnbelkinia, Aedes, Psorophora, Mansonia and Coquillettidia) and to 5 Tribes out of the 9 present in the country. The Aedini, Sabethini and Culicini Tribes were richer in species with 5, 4 and 4 species, respectively, than the Mansonini (2 species) and Toxorhynchitini (1 species) Tribes. We discuss some bioecological aspects regarding the 16 new-species records in Falcon State and give a checklist of the mosquito species previously reported in the literature.