Recent advances enabling the cloning of human immunoglobulin G genes have proven effective for discovering monoclonal antibodies with therapeutic potential. However, these antibody-discovery methods are often arduous and identify only a few candidates from numerous antibody-secreting plasma cells or plasmablasts. We describe an in vivo enrichment technique that identifies broadly neutralizing human antibodies with high frequency. For this technique, human peripheral blood mononuclear cells from vaccinated donors are activated and enriched in an antigen-specific manner for the production of numerous antigen-specific plasmablasts. Using this technology, we identified four broadly neutralizing influenza A antibodies by screening only 840 human antibodies. Two of these antibodies neutralize every influenza A human isolate tested and perform better than the current anti-influenza A therapeutic, oseltamivir, in treating severe influenza infection in mice and ferrets. Furthermore, these antibodies elicit robust in vivo synergism when combined with oseltamivir, thus highlighting treatment strategies that could benefit influenza-infected patients.

Folate receptor alpha (FR␣) has been described as a factor involved in mediating Ebola virus entry into cells (6). Furthermore, it was suggested that interaction with FR␣ results in internalization and subsequent viral ingress into the cytoplasm via caveolae (9). Descriptions of cellular receptors for Ebola virus and its entry mechanisms are of fundamental importance, particularly with the advent of vectors bearing Ebola virus glycoprotein (GP) being utilized for gene transfer into cell types such as airway epithelial cells. Thus, the ability of FR␣ to mediate efficient entry of viral pseudotypes carrying GP was investigated. We identified cell lines and primary cell types such as macrophages that were readily infected by GP pseudotypes despite lacking detectable surface FR␣, indicating that this receptor is not essential for Ebola virus infection. Furthermore, we find that T-cell lines stably expressing FR␣ are not infectible, suggesting that FR␣ is also not sufficient to mediate entry. T-cell lines lack caveolae, the predominant route of FR␣-mediated folate metabolism. However, the coexpression of FR␣ with caveolin-1, the major structural protein of caveolae, was not able to rescue infectivity in a T-cell line. In addition, other cell types lacking caveolae are fully infectible by GP pseudotypes. Finally, a panel of ligands to and soluble analogues of FR␣ were unable to inhibit infection on a range of cell lines, questioning the role of FR␣ as an important factor for Ebola virus entry.

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Broadly neutralizing antibodies targeting the stalk region of influenza A virus (IAV) hemagglutinin (HA) are effective in blocking virus infection both in vitro and in vivo. The highly conserved epitopes recognized by these antibodies are critical for the membrane fusion function of HA and therefore less likely to be permissive for virus mutational escape. Here we report three resistant viruses of the A/Perth/16/2009 strain that were selected in the presence of a broadly neutralizing stalk-binding antibody. The three resistant viruses harbor three different mutations in the HA stalk: (1) Gln387Lys; (2) Asp391Tyr; (3) Asp391Gly. The Gln387Lys mutation completely abolishes binding of the antibody to the HA stalk epitope. The other two mutations, Asp391Tyr and Asp391Gly, do not affect antibody binding at neutral pH and only slightly reduce binding at low pH. Interestingly, they enhance the fusion ability of the HA, representing a novel mechanism that allows productive membrane fusion even in the presence of antibody and hence virus escape from antibody neutralization. Therefore, these mutations illustrate two different resistance mechanisms used by IAV to escape broadly neutralizing stalk-binding antibodies. Compared to the wild type virus, the resistant viruses release fewer progeny viral particles during replication and are more sensitive to Tamiflu, suggesting reduced viral fitness.

Elevated cathepsin F and the ability of its inhibitor to produce a more normal phenotype in diabetic corneas suggest increased proteolysis in these corneas. Proteinase changes may result from abnormalities of growth factors, such as HGF and FGF-3, in DR corneas. Specific modulation of proteinases and growth factors could reduce diabetic corneal epitheliopathy.

In the sera of patients infected with hepatitis B virus (HBV), in addition to infectious particles, there is an excess (typically 1,000-to 100,000-fold) of empty subviral particles (SVP) composed solely of HBV envelope proteins in the form of relatively smaller spheres and filaments of variable length. Hepatitis delta virus (HDV) assembly also uses the envelope proteins of HBV to produce an infectious particle. Rate-zonal sedimentation was used to study the particles released from liver cell lines that produced SVP only, HDV plus SVP, and HBV plus SVP. The SVP made in the absence of HBV or HDV were further examined by electron microscopy. They bound efficiently to heparin columns, consistent with an ability to bind cell surface glycosaminoglycans. However, unlike soluble forms of HBV envelope protein that were potent inhibitors, the SVP did not inhibit the ability of HBV and HDV to infect primary human hepatocytes.In natural infections with hepatitis B virus (HBV), there is an excess of empty noninfectious subviral particles (SVP) that do not contain the viral capsid. SVP are typically present in a 1,000-to 100,000-fold excess relative to the infectious particles (12, 13). They exist in two main forms: spheres of 25 nm in diameter and filaments of 22 nm in diameter with variable length (15,17). They can contain all three forms of the HBV envelope proteins: L, M, and S. These share a common C terminus, with M containing the pre-S2 domain relative to S and L containing the pre-S1 domain relative to M (15). There is good evidence that during infection a domain within the pre-S1 of L is what interacts with an as-yet-unidentified host receptor(s) (15). Hepatitis delta virus (HDV), which is assembled using the envelope proteins of HBV, also depends upon this pre-S1 domain (26). HDV can be assembled using only the S protein of HBV, but the particles are noninfectious.SVP from patients are immunogenic and were used with success in the first HBV vaccine (1). Most current HBV vaccines are prepared in yeast from SVP assembled using just the HBV S protein; such SVP are sufficient to protect individuals against HBV and HDV.The basis for the excess of SVP detected in patients is unexplained, and the biological function of this excess has been largely ignored. Some authors have suggested that SVP might sop up neutralizing antibodies produced by the host and thus increase the ability of the infectious particles to reach susceptible cells (11,25). It has also been suggested that SVP contribute to a state of immune tolerance that is a precondition for highly productive persistent infection (13). One study with SVP of duck HBV indicated that for infections at low multiplicity SVP could enhance infection, but when present in large amounts they were inhibitory (3). Another study showed that SVP containing the large envelope protein interfered with duck HBV infection (19).For the present studies we chose to use SVP as produced by transfection procedures. For the following reasons we consider these more defined than SVP obtained from ...

Subgroup J avian leukosis virus (ALV-J) is a recently identified avian oncogenic retrovirus responsible for severe economic losses worldwide. In contrast with the other ALV subgroups, ALV-J predominantly induces myeloid leukosis in meat-type chickens. Despite significant homology with the other ALV subgroups across most of the genome, the envelope protein of ALV-J (EnvJ) shares low homology with the others. Pathogenicity and myeloid leukosis induction map to the env gene of ALV-J. A chimeric protein composed of the surface domain of EnvJ fused to the constant region of a rabbit IgG and mass spectrometry were used to identify the chicken Na ؉ ͞H ؉ exchanger type 1 (chNHE1) as a binding protein for ALV-J. Flow cytometry analysis and coprecipitation experiments demonstrated a specific interaction between EnvJ and chNHE1. When introduced into nonpermissive human 293T cells and quail QT6 cells, chNHE1 conferred susceptibility to EnvJ-mediated infection. Furthermore, 293T cells expressing chNHE1 fused with 293T cells expressing EnvJ in a low-pH-dependent manner. Together, these data identify chNHE1 as a cellular receptor for the highly pathogenic ALV-J.retrovirus ͉ viral receptor ͉ viral envelope A vian leukosis viruses (ALV) are a group of avian retroviruses that induce tumors in host birds. The viruses that infect chickens are classified into six subgroups (A-E and J) on the basis of the envelope glycoprotein responsible for specific viral interference patterns, virus neutralization, and host range (1). The most recently identified subgroup, ALV-J, predominantly infects meattype chickens and turkeys (1). ALV-J was identified in 1988 and became widespread in commercial meat-type poultry during the 1990s. The transmission of ALV-J is much higher than other ALV subgroups (2), thus making control and eradication significantly more difficult. The virus also evolves rapidly with sequence variations clustered in hypervariable regions of the envelope protein (Env) (3). In contrast to other subgroups, which primarily cause lymphoma, ALV-J mainly induces myeloid leukosis (4). ALV-J infection causes disease and death in both broiler breeders and egg layers and represents a significant problem for the commercial poultry industry worldwide, with estimated losses of 1.5% per week in excess mortality (5).Retroviruses infect host cells through specific interactions between viral Env and cell surface receptors. The Env surface subunit (SU) directly binds to the receptor, and subsequent conformational changes in the Env transmembrane (TM) subunit drive fusion of the viral and cellular membranes (6). The receptors for all of the other major ALV subgroups (A-E) have been identified (7-10). Receptor distribution is a major determinant of ALV subgroup tropism. Env is also a major determinant for the induction of lymphoid and myeloid tumors by ALV-A and ALV-J, respectively (4), presumably because the specific receptors for ALV-A and ALV-J are differentially expressed on different cell lineages. This hypothesis is supported by the observat...

The DAZ (Deleted in AZoospermia) and DAZLA (DAZ-like autosomal) genes may be determinants of male infertility. The DAZ gene on the long arm of the human Y chromosome is a strong candidate for the 'azoospermia factor' (AZF). Its role in spermatogenesis is supported by its exclusive expression in testis, its deletion in a high percentage of males with azoospermia or severe oligospermia, and its homology with a Drosophila male infertility gene boule. No DAZ homologous sequences have been found on the mouse Y chromosome. Instead, a Dazla gene was isolated from mouse chromosome 17 and has been considered to be a murine homologue of DAZ. However, the homology between human DAZ and mouse Dazla is not strong, and Dazla contains only one of the seven DAZ repeats found in DAZ. We report the isolation of the human DAZLA gene by screening a human testis cDNA library with a DAZ cDNA clone. DAZLA encodes only one DAZ repeat and shares high homology with the mouse Dazla, indicating that these two genes are homologues. Using a panel of rodent-human somatic cell lines and fluorescence in situ hybridization, the DAZLA gene was mapped to 3p24, a region not known to share homology with mouse chromosome 17. The DAZLA gene may be involved in some familial cases of autosomal recessive male infertility.