Production of antibodies can last for a lifetime, through mechanisms that remain poorly understood. Here, we show that human memory B lymphocytes proliferate and differentiate into plasma cells in response to polyclonal stimuli, such as bystander T cell help and CpG DNA. Furthermore, plasma cells secreting antibodies to recall antigens are produced in vivo at levels proportional to the frequency of specific memory B cells, even several years after antigenic stimulation. Although antigen boosting leads to a transient increase in specific antibody levels, ongoing polyclonal activation of memory B cells offers a means to maintain serological memory for a human lifetime.

Toll-like receptors (TLRs) are pattern recognition receptors that trigger innate immunity. In this study we investigated the expression of 10 TLRs in human naive and memory B-cell subsets. We report that in human naive B cells most TLRs are expressed at low to undetectable levels, but the expression of TLR9 and TLR10 is rapidly induced following B-cell-receptor ( IntroductionToll-like receptors (TLRs) are pattern recognition receptors that trigger innate immunity, providing both immediate protective responses against pathogens and instructing the adaptive immune response through the induction of dendritic cell recruitment and maturation. 1-4 TLR triggering results in nuclear factor kappa B (NF-B)-mediated activation of inflammatory genes. 5 Ten TLRs have been described in humans, and agonists have been defined for 9 of them. 6 TLRs can function as homodimers or heterodimers, increasing the repertoire of specificities. 7 TLR1, TLR2, and TLR6 are triggered by peptidoglycan and other microbial products, 8,9 TLR3 by double-stranded RNA, 10 TLR4 by lipopolysaccharide (LPS), 11 TLR5 by flagellin, 12 TLR7 and TLR8 by imidazoquinolines, 6,13 and TLR9 by unmethylated CpG DNA. [14][15][16][17][18][19][20][21] TLRs are differentially expressed in dendritic cell (DC) subsets. [22][23][24] Monocytes and myeloid DCs express TLR2 and TLR4 and respond to peptidoglycan and LPS, while plasmacytoid DCs express a reciprocal pattern-namely, TLR7 and TLR9 and respond to CpG. The induction of DC maturation by TLRs represents the functional link between innate and adaptive response. 1,25 TLR expression can be regulated; for instance, interferon-␥ (IFN-␥) up-regulates TLR4 expression in human phagocytes, enhancing their capacity to respond to LPS. 26 Mouse B cells proliferate and differentiate in response to LPS or CpG, 14,27 indicating that they express functional TLR4 and TLR9. Human B cells also respond to CpG, 19 but recent data indicate that memory B cells are more responsive than naive B cells, raising the possibility that TLR expression may be differentially regulated in human naive and memory B cells. 28 We report here that in human naive B cells TLRs are expressed at low to undetectable levels but their expression is rapidly up-regulated by B-cell-receptor (BCR) triggering. In contrast, memory B cells express several TLRs at constitutively high levels. The differential expression correlates with responsiveness to CpG DNA, an agonist of TLR9, and points to a relevant difference between human and mouse B cells. Thus, in humans TLRs are downstream of BCR and play a role in the late phase of acquired immunity. Materials and methods Cell isolation and cultureB lymphocytes were isolated from peripheral blood mononuclear cells (PBMCs) using CD19 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and further purified by cell sorting using a FACSVantage (Becton Dickinson, San Jose, CA). Cells were stained with directly labeled antibodies to CD27 (Pharmingen, San Diego, CA) (to identify memory B cells) and appropriate combinations...

Human cytomegalovirus (HCMV) is a widely circulating pathogen that causes severe disease in immunocompromised patients and infected fetuses. By immortalizing memory B cells from HCMV-immune donors, we isolated a panel of human monoclonal antibodies that neutralized at extremely low concentrations (90% inhibitory concentration [IC 90 ] values ranging from 5 to 200 pM) HCMV infection of endothelial, epithelial, and myeloid cells. With the single exception of an antibody that bound to a conserved epitope in the UL128 gene product, all other antibodies bound to conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex. Antibodies against gB, gH, or gM/gN were also isolated and, albeit less potent, were able to neutralize infection of both endothelial-epithelial cells and fibroblasts. This study describes unusually potent neutralizing antibodies against HCMV that might be used for passive immunotherapy and identifies, through the use of such antibodies, novel antigenic targets in HCMV for the design of immunogens capable of eliciting previously unknown neutralizing antibody responses.Human cytomegalovirus (HCMV) is a member of the herpesvirus family which is widely distributed in the human population and can cause severe disease in immunocompromised patients and upon infection of the fetus. HCMV infection causes clinical disease in 75% of patients in the first year after transplantation (58), while primary maternal infection is a major cause of congenital birth defects including hearing loss and mental retardation (5,33,45). Because of the danger posed by this virus, development of an effective vaccine is considered of highest priority (51).HCMV infection requires initial interaction with the cell surface through binding to heparan sulfate proteoglycans (8) and possibly other surface receptors (12,23,64,65). The virus displays a broad host cell range (24, 53), being able to infect several cell types such as endothelial cells, epithelial cells (including retinal cells), smooth muscle cells, fibroblasts, leukocytes, and dendritic cells (21,37,44,54). Endothelial cell tropism has been regarded as a potential virulence factor that might influence the clinical course of infection (16, 55), whereas infection of leukocytes has been considered a mechanism of viral spread (17,43,44). Extensive propagation of HCMV laboratory strains in fibroblasts results in deletions or mutations of genes in the UL131A-128 locus (1,18,21,36,62,63), which are associated with the loss of the ability to infect endothelial cells, epithelial cells, and leukocytes (15,43,55,61). Consistent with this notion, mouse monoclonal antibodies (MAbs) to UL128 or UL130 block infection of epithelial and endothelial cells but not of fibroblasts (63). Recently, it has been shown that UL128, UL130, and UL131A assemble with gH and gL to form a five-protein complex (thereafter designated gH/gL/UL128-131A) that is an alternative to the previously described gCIII complex made of gH, gL, and gO (22,28,48,63).In immunoco...

BackgroundNew prophylactic and therapeutic strategies to combat human infections with highly pathogenic avian influenza (HPAI) H5N1 viruses are needed. We generated neutralizing anti-H5N1 human monoclonal antibodies (mAbs) and tested their efficacy for prophylaxis and therapy in a murine model of infection.Methods and FindingsUsing Epstein-Barr virus we immortalized memory B cells from Vietnamese adults who had recovered from infections with HPAI H5N1 viruses. Supernatants from B cell lines were screened in a virus neutralization assay. B cell lines secreting neutralizing antibodies were cloned and the mAbs purified. The cross-reactivity of these antibodies for different strains of H5N1 was tested in vitro by neutralization assays, and their prophylactic and therapeutic efficacy in vivo was tested in mice. In vitro, mAbs FLA3.14 and FLD20.19 neutralized both Clade I and Clade II H5N1 viruses, whilst FLA5.10 and FLD21.140 neutralized Clade I viruses only. In vivo, FLA3.14 and FLA5.10 conferred protection from lethality in mice challenged with A/Vietnam/1203/04 (H5N1) in a dose-dependent manner. mAb prophylaxis provided a statistically significant reduction in pulmonary virus titer, reduced associated inflammation in the lungs, and restricted extrapulmonary dissemination of the virus. Therapeutic doses of FLA3.14, FLA5.10, FLD20.19, and FLD21.140 provided robust protection from lethality at least up to 72 h postinfection with A/Vietnam/1203/04 (H5N1). mAbs FLA3.14, FLD21.140 and FLD20.19, but not FLA5.10, were also therapeutically active in vivo against the Clade II virus A/Indonesia/5/2005 (H5N1).ConclusionsThese studies provide proof of concept that fully human mAbs with neutralizing activity can be rapidly generated from the peripheral blood of convalescent patients and that these mAbs are effective for the prevention and treatment of H5N1 infection in a mouse model. A panel of neutralizing, cross-reactive mAbs might be useful for prophylaxis or adjunctive treatment of human cases of H5N1 influenza.

Pregnancy-associated malaria (PAM) is caused by Plasmodium falciparum-infected erythrocytes (IEs) that bind to chondroitin sulphate A (CSA) in the placenta by PAM-associated clonally variant surface antigens (VSA). Pregnancy-specific VSA (VSAPAM), which include the PfEMP1 variant VAR2CSA, are targets of IgG-mediated protective immunity to PAM. Here, we report an investigation of the specificity of naturally acquired immunity to PAM, using eight human monoclonal IgG1 antibodies that react exclusively with intact CSA-adhering IEs expressing VSAPAM. Four reacted in Western blotting with high-molecular-weight (> 200 kDa) proteins, while seven reacted with either the DBL3-X or the DBL5-ε domains of VAR2CSA expressed either as Baculovirus constructs or on the surface of transfected Jurkat cells. We used a panel of recombinant antigens representing DBL3-X domains from P. falciparum field isolates to evaluate B-cell epitope diversity among parasite isolates, and identified the binding site of one monoclonal antibody using a chimeric DBL3-X construct. Our findings show that there is a high-frequency memory response to VSAPAM, indicating that VAR2CSA is a primary target of naturally acquired PAM-specific protective immunity, and demonstrate the value of human monoclonal antibodies and conformationally intact recombinant antigens in VSA characterization.