The rapid spread of highly pathogenic avian influenza (HPAI) H5N1 virus underscores the importance of effective antiviral treatment. Previously, we developed human monoclonal antibodies 65C6 and 100F4 that neutralize almost all (sub)clades of HPAI H5N1. The conserved 65C6 epitope was mapped to the globular head of HA. However, neither the 100F4 epitope nor the neutralization mechanism by these antibodies was known. In this study, we determined the 100F4 epitope and unraveled a neutralization mechanism by antibodies 65C6 and 100F4. Influenza A virus infection is a persistent threat to public health worldwide. High-affinity neutralizing antibodies against conserved epitopes could provide immunity to diverse influenza virus strains and protection against future pandemic viruses. Previously, we developed human monoclonal antibodies (MAb) 65C6 and 100F4 that potently neutralize all H5 clades and subclades of highly pathogenic avian influenza (HPAI) H5N1 viruses except subclade 7.2 (Fig. 1A) and defined a conformational 65C6 epitope (1). In this study, we determined the 100F4 epitope and dissected the neutralization mechanism by these antibodies.To map the 100F4 epitope, a yeast display analysis was carried out similarly to the way we mapped the 65C6 epitope (1, 2). Figure 1B shows the 15 single amino acid mutations in H5 hemagglutinin (HA) that abolish the binding of antibody 100F4. Among these, the 7 residues at positions 68,112, 137, 143, 251, 254, and 255 were on the HA surface, while the rest were underneath the surface.To test whether these 7 surface mutations would affect neutralization by antibody 100F4, genes encoding 7 full-length H5 HA single mutants derived from H5N1 strain A/Beijing/01/03 subclade 7.1 were constructed and used to generate H5N1 pseudotypes. The resistance of H5N1 pseudotypes to antibody 100F4 was measured with the pseudotype-based neutralization assay (3). Compared to the wild-type subclade 7.1 H5N1 pseudotype, only H5N1 pseudotypes expressing H5 HA mutants with mutations at position 68 or 112 (72 or 116 according to H3 numbering) were dramatically resistant to antibody 100F4 (Fig. 1C and D). On the HA surface, these two resistant residues are adjacent to each other (Fig. 1E), but they are next to the Cb in H1 HA and site E in H3 HA (4-7) ( Fig. 1F and G). The 100F4 epitope does not overlap any known epitopes in the head region detected by human and mouse MAb ( Fig. 1H and I). Thus, the 100F4 epitope is a new conserved conformational epitope on the globular head and away from the receptor binding site (RBS). In contrast, the 65C6 epitope partially overlaps with Sa in H1 HA at residue 161 (K165 according to H3 numbering) and with site A in H3 HA at residues 118 and 121 (T122 and F125 according to H3 numbering) (4-7). In addition, the 65C6 epitope also partially overlaps epitopes detected by some human MAb, i.e., FLA5.10 at P118 (P122 according to H3 numbering), FLD21.140 at S121, Y164, and T167 (S125, Y168, and T171 according to H3 numbering) (8), AVFLuigG01 at P118, Y164, and T167 (P122, ...

PG9 and PG16 are two recently isolated quaternary-specific human monoclonal antibodies that neutralize 70 to 80% of circulating HIV-1 isolates. The crystal structure of PG16 shows that it contains an exceptionally long CDR H3 that forms a unique stable subdomain that towers above the antibody surface to confer fine specificity. To determine whether this unique architecture of CDR H3 itself is sufficient for epitope recognition and neutralization, we cloned CDR H3 subdomains derived from human monoclonal antibodies PG16, PG9, b12, E51, and AVF and genetically linked them to a glycosyl-phosphatidylinositol (GPI) attachment signal. Each fusion gene construct is expressed and targeted to lipid rafts of plasma membranes through a GPI anchor. Moreover, GPI-CDR H3(PG16, PG9, and E51), but not GPI-CDR H3(b12 and AVF), specifically neutralized multiple clades of HIV-1 isolates with a great degree of potency when expressed on the surface of transduced TZM-bl cells. Furthermore, GPI-anchored CDR H3(PG16), but not GPI-anchored CDR H3(AVF), specifically confers resistance to HIV-1 infection when expressed on the surface of transduced human CD4؉ T cells. Finally, the CDR H3 mutations (Y100HF, D100IA, and G7) that were previously shown to compromise the neutralization activity of antibody PG16 also abolished the neutralization activity of GPI-CDR H3(PG16). Thus, we conclude that the CDR H3 subdomain of PG16 neutralizes HIV-1 when targeted to the lipid raft of the plasma membrane of HIV-1-susceptible cells and that GPI-CDR H3 can be an alternative approach for determining whether the CDR H3 of certain antibodies alone can exert epitope recognition and neutralization.During human immunodeficiency virus type 1 (HIV-1) infection, a proportion of individuals develop broadly neutralizing sera over time (32). From a few such individuals, a number of potent and broadly cross-neutralizing monoclonal antibodies (MAbs) have also been isolated (36,38,40). Among them, PG9 and PG16 are recently isolated quaternary-specific neutralizing MAbs from a subtype A HIV-1-infected individual in Africa that neutralize 70 to 80% of circulating HIV-1 isolates (36). PG9 and PG16 bind to overlapping, but distinct, gp120 epitopes composed of conserved elements from the second and third variable regions (V2 and V3, respectively). The quaternary epitopes are glycosylated (6) and are preferentially displayed on envelope trimers on the surface of virions and transfected cells but not on recombinant monomeric gp120 or soluble trimers (36).To gain insight into the molecular features of antibody binding and neutralizing activities, Pancera et al. (23) and Pejchal et al. (24) recently determined the crystal structures of the Fab fragment of PG16. Antibodies PG9 and PG16 were found to be sulfated (24). The fine specificity of the antibodies is conferred by an exceptionally long third-heavy-chain complementarity-determining region (CDR H3) that forms a unique stable subdomain towering above the antibody surface (23, 24).

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How overall tumor growth emerges from the properties of functionally heterogeneous tumor cell subpopulations is a fundamental question of cancer biology. Here we combined lineage tracing, continuous monitoring of tumor mass, proliferation assays and transcriptomics with mathematical modeling and statistical inference to dissect the growth of glioblastoma in mice. We found that tumors grow exponentially at the rate of symmetric divisions of brain tumor stem cells (BTSCs). Spatial modeling predicts, and data show, that BTSCs accumulate at the tumor rim rather than in the core. The physiological differentiation hierarchy downstream of BTSCs is preserved in mice and humans: transit amplifying progenitors give rise to terminally differentiated cells. Consistent with our quantification of the mechanisms underlying tumor growth, molecular data show elevated expression of cell cycle-and migration-related genes in BTSCs. Our systematic approach reveals fundamental properties of glioblastoma and may be transferable to the study of other animal models of cancer.