The amyloid-β lowering capacity of anti-Aβ antibodies has been demonstrated in transgenic models of Alzheimer's disease (AD) and in AD patients. While the mechanism of immunotherapeutic amyloid-β removal is controversial, antibody-mediated sequestration of peripheral Aβ versus microglial phagocytic activity and disassembly of cerebral amyloid (or a combination thereof) has been proposed. For successful Aβ immunotherapy, we hypothesized that high affinity antibody binding to amyloid-β plaques and recruitment of brain effector cells is required for most efficient amyloid clearance. Here we report the generation of a novel fully human anti-Aβ antibody, gantenerumab, optimized in vitro for binding with sub-nanomolar affinity to a conformational epitope expressed on amyloid-β fibrils using HuCAL(®) phage display technologies. In peptide maps, both N-terminal and central portions of Aβ were recognized by gantenerumab. Remarkably, a novel orientation of N-terminal Aβ bound to the complementarity determining regions was identified by x-ray analysis of a gantenerumab Fab-Aβ(1-11) complex. In functional assays gantenerumab induced cellular phagocytosis of human amyloid-β deposits in AD brain slices when co-cultured with primary human macrophages and neutralized oligomeric Aβ42-mediated inhibitory effects on long-term potentiation in rat brain. In APP751(swedish)xPS2(N141I) transgenic mice, gantenerumab showed sustained binding to cerebral amyloid-β and, upon chronic treatment, significantly reduced small amyloid-β plaques by recruiting microglia and prevented new plaque formation. Unlike other Aβ antibodies, gantenerumab did not alter plasma Aβ suggesting undisturbed systemic clearance of soluble Aβ. These studies demonstrated that gantenerumab preferentially interacts with aggregated Aβ in the brain and lowers amyloid-β by eliciting effector cell-mediated clearance.

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A recombinant baculovirus expressing glycoprotein H (gpUL75) of human cytomegalovirus was used to examine the humoral immune response in naturally infected individuals. Recombinant baculovirus infected insect cells produced two forms of gH with molecular masses of 78-82 kDa and 94 kDa. The 94 kDa polypeptide was modified by high mannose oligosaccharide side-chains as shown by reduction in molecular mass after treatment with endoglycosidases H and F. The 78-82 kDa protein represented the non-glycosylated precursor which was resistant to the enzymes. In contrast to gH expressed in mammalian cells, the recombinant baculovirus expressed gH was transported to the cell surface. Glycoprotein H produced in insect cells was reactive with human convalescent sera and all tested neutralizing monoclonal antibodies recognizing either linear or conformational epitopes. Antibodies reacting with insect cell derived gH were detected in 96% of HCMV seropositive human sera. Using insect cells infected with the gH expressing recombinant baculovirus as immunoabsorbent, between 0% and 58% of the total virus neutralizing activity was removed from sera of individuals with a past HCMV infection, gH must therefore be considered a major antigen for the induction of neutralizing antibodies during natural infection.

Bacterial fusion proteins, constructed from overlapping fragments of the open reading frame coding for gp86 of human cytomegalovirus (HCMV) strain AD169, were used to localize antigenic regions recognized by antibodies from human convalescent sera. A major domain for binding of conformation-independent antibodies was localized on fusion protein AP86, containing amino acids 15 to 142 of gp86. Human antibodies, affinity purified on AP86, neutralized infectious virus in tissue culture. In addition, a mouse monoclonal antibody (AP86-SA4), raised against AP86, also neutralized HCMV. AP86-SA4 was reactive with viral gp86 in immunoblot assays and showed a plasma membrane staining on intact HCMV-infected fibroblasts late in infection. After exonuclease III deletions of the viral gene, the binding site of neutralizing human as well as mouse antibodies was localized between amino acid residues 34 and 43. The domain has sequence variation between laboratory strains AD169 and Towne, and binding of the antibodies was strain specific. To our knowledge, this is the first characterization of a strain-specific neutralizing epitope on HCMV.

The IgG subclass pattern against linear antibody binding sites on glycoproteins of human cytomegalovirus (HCMV) was investigated in HCMV-positive healthy blood donors, human immunodeficiency virus-infected persons, sera from mothers with primary HCMV infection during pregnancy and their children, and sequential sera from transplant recipients. As antigens, three immunodominant domains capable of inducing neutralizing antibodies during natural infection were selected on glycoproteins gp58/116 (gB) and gp86 (gH). Bacterial fusion proteins representing these regions were used as antigens in a subclass-specific ELISA. Reactivity against the antibody binding site on gp86 was detected in both the IgG1 and IgG3 subclasses. In contrast, exclusively IgG1 antibodies were found against both linear domains on glycoprotein complex gp58/116 and also against full-length gp58/116 expressed in insect cells. The data demonstrate a differential regulation of the antibody response to envelope components of HCMV.