HA Antibody-Mediated FcγRIIIa Activity Is Both Dependent on FcR Engagement and Interactions between HA and Sialic Acids.

Interactions with receptors for the Fc region of IgG (FcγRs) have been shown to contribute to the in vivo protection against influenza A viruses provided by broadly neutralizing antibodies (bnAbs) that bind to the viral hemagglutinin (HA) stem. In particular, Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) has been shown to contribute to protection by stem-binding bnAbs. Fc-mediated effector functions appear not to contribute to protection provided by strain-specific HA head-binding antibodies. We used a panel of anti-stem and anti-head influenza A and B monoclonal antibodies with identical human IgG1 Fc domains and investigated their ability to mediate ADCC-associated FcγRIIIa activation. Antibodies which do not interfere with sialic acid binding of HA can mediate FcγRIIIa activation. However, the FcγRIIIa activation was inhibited when a mutant HA, unable to bind sialic acids, was used. Antibodies which block sialic acid receptor interactions of HA interfered with FcγRIIIa activation. The inhibition of FcγRIIIa activation by HA head-binding and sialic acid receptor-blocking antibodies was confirmed in plasma samples of H5N1 vaccinated human subjects. Together, these results suggest that in addition to Fc-FcγR binding, interactions between HA and sialic acids on immune cells are required for optimal Fc-mediated effector functions by anti-HA antibodies.

Highly conserved protective epitopes on influenza B viruses.

Identification of broadly neutralizing antibodies against influenza A viruses has raised hopes for the development of monoclonal antibody-based immunotherapy and "universal" vaccines for influenza. However, a substantial part of the annual flu burden is caused by two cocirculating, antigenically distinct lineages of influenza B viruses. Here, we report human monoclonal antibodies, CR8033, CR8071, and CR9114, that protect mice against lethal challenge from both lineages. Antibodies CR8033 and CR8071 recognize distinct conserved epitopes in the head region of the influenza B hemagglutinin (HA), whereas CR9114 binds a conserved epitope in the HA stem and protects against lethal challenge with influenza A and B viruses. These antibodies may inform on development of monoclonal antibody-based treatments and a universal flu vaccine for all influenza A and B viruses.

Novel replication-incompetent vector derived from adenovirus type 11 (Ad11) for vaccination and gene therapy: low seroprevalence and non-cross-reactivity with Ad5.

A novel plasmid-based adenovirus vector system that enables manufacturing of replication-incompetent (DeltaE1) adenovirus type 11 (Ad11)-based vectors is described. Ad11 vectors are produced on PER.C6/55K cells yielding high-titer vector batches after purification. Ad11 seroprevalence proves to be significantly lower than that of Ad5, and neutralizing antibody titers against Ad11 are low. Ad11 seroprevalence among human immunodeficiency virus-positive (HIV(+)) individuals is as low as that among HIV(-) individuals, independent of the level of immune suppression. The low level of coinciding seroprevalence between Ad11 and Ad35 in addition to a lack of correlation between high neutralizing antibody titers towards either adenovirus strongly suggest that the limited humoral cross-reactive immunity between these two highly related B viruses appears not to preclude the use of both vectors in the same individual. Ad11 transduces primary cells including smooth muscle cells, synoviocytes, and dendritic cells and cardiovascular tissues with higher efficiency than Ad5. Ad11 and Ad35 appear to have a similar tropism as judged by green fluorescent protein expression levels determined by using a panel of cancer cell lines. In addition, Ad5 preimmunization did not significantly affect Ad11-mediated transduction in C57BL/6 mice. We therefore conclude that the Ad11-based vector represents a novel and useful candidate gene transfer vehicle for vaccination and gene therapy.

Spectrum of genetic changes in gastro-esophageal cancer cell lines determined by an integrated molecular cytogenetic approach.

Adenocarcinomas arising around the gastro-esophageal junction (GEJ) are highly malignant, and their incidence has risen rapidly in the last decades. Cell lines are the basic in vitro system for functional and therapeutic studies in GEJ tumors, but only a small number of cell lines are currently available, and none of them has been fully karyotyped. We analyzed 5 GEJ tumor cell lines using a combination of 24-color fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH) and genomic microarrays. Using CGH we demonstrated that these cell lines present imbalances similar to those we had previously observed in primary GEJ tumors, namely gains on 1q, 7q, 8q, 17q, 19q, 20, and X, and losses on 3p, 4, 5q, 9p, 18q, and 21. Multicolor FISH karyotyping revealed multiple structural rearrangements involving chromosomes 1, 5, 6, 7, 8, 9, 13, 17, 18, and 22. Rearrangements of chromosome 8 involved 10 different chromosomes, while rearrangements of chromosome 17 involved 5. Different rearrangements resulted in imbalances of similar chromosome regions, suggesting that similar genomic imbalances are constitutively important but are achieved through different pathways. The use of a commercially available genomic array excluded TOP2A (17q), and MYBL2, PTPT1, CSE1L, and ZNF217 (20q) as candidate genes for frequently amplified areas on these chromosomes, and contributed to refining the limits of chromosome regions involved in genomic imbalances.