Generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune B cells

Funaro, Ada; Gribaudo, Giorgio; Luganini, Anna; Ortolan, Erika; Buono, Nicola Lo; Vicenzi, Elisa; Cassetta, Luca; Landolfo, Santo; Buick, Richard; Falciola, Luca; Murphy, Marianne; Garotta, Gianni; Malavasi, Fabio

doi:10.1186/1472-6750-8-85

Cited by 21 publications

(21 citation statements)

References 55 publications

(57 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For example, the anti-respiratory syncytial virus mAb palivizumab and the anti-coronavirus mAb CR3014 have an IC 50 of 2 mg/ml (29,30), whereas the reported IC 50 of anti-influenza virus, antihuman CMV, and anti-West Nile virus mAbs range between 55 and 92 ng/ml (31)(32)(33). Therefore, the in vitro potency of 5F10 and 8B10 might be adequate for in vivo protection, which remains to be tested.…”

Section: The Journal Of Immunologymentioning

confidence: 99%

Chikungunya Virus Envelope-Specific Human Monoclonal Antibodies with Broad Neutralization Potency

Warter

Lee

Thiagarajan

et al. 2011

The Journal of Immunology

View full text Add to dashboard Cite

Chikungunya virus (CHIKV) is an alphavirus responsible for numerous epidemics in Africa and Asia. Infection by CHIKV is often characterized by long-lasting, incapacitating arthritis, and some fatal cases have been described among elderly and newborns. Currently, there is no available vaccine or specific treatment against CHIKV. Blood B cells from a donor with history of CHIKV infection were activated, immortalized, amplified, and cloned. Two human mAbs against CHIKV, 5F10 and 8B10, were identified, sequenced, and expressed in recombinant form for characterization. In a plaque reduction neutralization test, 5F10 and 8B10 show mean IC50 of 72 and 46 ng/ml, respectively. Moreover, both mAbs lead to a strong decrease in extracellular spreading of infectious viral particles from infected to uninfected cells. Importantly, the mAbs neutralize different CHIKV isolates from Singapore, Africa, and Indonesia, as well as O’nyong-nyong virus, but do not recognize other alphaviruses tested. Both mAbs are specific for the CHIKV envelope: 5F10 binds to the E2 glycoprotein ectodomain and 8B10 to E1 and/or E2. In conclusion, these two unique human mAbs strongly, broadly, and specifically neutralize CHIKV infection in vitro and might become possible therapeutic tools against CHIKV infection, especially in individuals at risk for severe disease. Importantly, these mAbs will also represent precious tools for future studies on host–pathogen interactions and the rational design of vaccines against CHIKV.

show abstract

Section: The Journal Of Immunologymentioning

confidence: 99%

Chikungunya Virus Envelope-Specific Human Monoclonal Antibodies with Broad Neutralization Potency

Warter

Lee

Thiagarajan

et al. 2011

The Journal of Immunology

View full text Add to dashboard Cite

show abstract

“…CD22-positive memory B cells were purified by magnetic selection (Miltenyi Biotech) from peripheral blood mononuclear cells (PBMCs) obtained from 1 patient with diffuse SSc designated PAM (47-year-old woman with esophagus and lung involvement, a Rodnan skin thickness score of 17, positivity for antinuclear antibodies and anti-Scl-70 antibodies, and negativity for anticentromere antibodies) (8). These cells (designated the PAM B cells) were immortalized using Epstein-Barr virus, as described previously (9)(10)(11). Three days postinfection, the cells were manually counted and seeded (5 cells/well) in 96-well plates in the presence of irradiated (30 Gy) allogenic PBMCs.…”

Section: Methodsmentioning

confidence: 99%

“…Finally, direct and competitive ELISAs were performed with serum samples from 29 consecutive patients with SLE (SLE Disease Activity Index range [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. The distribution of PDGFRa binding in SLE serum was comparable to that in healthy control serum, as determined by direct ELISA with PDGFRa-His.…”

Section: Epitope Specificity Of Anti-pdgfra Autoantibodies In Sscmentioning

confidence: 99%

Epitope Specificity Determines Pathogenicity and Detectability of Anti–Platelet‐Derived Growth Factor Receptor α Autoantibodies in Systemic Sclerosis

Moroncini

Grieco

Nacci

et al. 2015

Arthritis & Rheumatology

Self Cite

View full text Add to dashboard Cite

Objective. To identify the epitopes recognized by autoantibodies targeting platelet-derived growth factor receptor a (PDGFRa) in systemic sclerosis (SSc) and develop novel assays for detection of serum antiPDGFRa autoantibodies.Methods. Epstein-Barr virus-immortalized B cells from 1 patient with SSc (designated PAM) were screened for expression of IgG binding to PDGFRa and induction of reactive oxygen species in fibroblasts. The variable regions of anti-PDGFRa IgG were cloned into an IgG expression vector to generate distinct recombinant human monoclonal autoantibodies (mAb), which were characterized by binding and functional assays. The epitopes of anti-PDGFRa recombinant human mAb were defined by molecular docking, surface plasmon resonance binding assays, screening of a conformational peptide library spanning the PDGFRa extracellular domains, and expression analyses of alanine-scanned PDGFRa mutants. Direct or competitive enzyme-linked immunosorbent assays were established to detect all serum anti-PDGFRa autoantibodies or, selectively, the agonistic ones.Results. Three types of anti-PDGFRa recombinant human mAb, with the same V H but distinct V L chains, were generated. Nonagonistic V H PAM-V k 13B8 recognized 1 linear epitope, whereas agonistic V H PAM-V l 16F4 and V H PAM-V k 16F4 recognized 2 distinct conformational epitopes. Serum anti-PDGFRa antibodies were detected in 66 of 70 patients with SSc, 63 of 130 healthy controls, 11 of 26 patients with primary Raynaud's phenomenon (RP), and 13 of 29 patients with systemic lupus erythematosus (SLE). Serum V H PAM-V k 16F4-like antibodies were found in 24 of 34 patients with SSc, but not in healthy controls, patients with primary RP, or patients with SLE. Peptides composing the V H PAM-V k 16F4 epitope inhibited PDGFRa signaling triggered by serum IgG from SSc patients.Conclusion. Agonistic anti-PDGFRa autoantibodies are enriched in SSc sera and recognize specific conformational epitopes that can be used to discriminate agonistic from nonagonistic antibodies and block PDGFRa signaling in patients with SSc.

show abstract

“…Using specific immunoabsorption to recombinant antigens and neutralization assays using fibroblasts as model target cells, it was estimated that 40 to 70% of the serum neutralizing activity is directed against gB (6). Other studies described human neutralizing antibodies specific for gB, gH, or gM/gN viral glycoproteins (6,14,26,29,34,41,52,60). Remarkably, we have recently shown that human sera exhibit a more-than-100-foldhigher potency in neutralizing infection of endothelial cells than infection of fibroblasts (20).…”

mentioning

confidence: 99%

Isolation of Human Monoclonal Antibodies That Potently Neutralize Human Cytomegalovirus Infection by Targeting Different Epitopes on the gH/gL/UL128-131A Complex

Macagno¹,

Bernasconi²,

Vanzetta³

et al. 2010

J Virol

308

370

View full text Add to dashboard Cite

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...

show abstract

Generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune B cells

Abstract: Background: Human monoclonal antibodies (mAbs) generated as a result of the immune response are likely to be the most effective therapeutic antibodies, particularly in the case of infectious diseases against which the immune response is protective.

Cited by 21 publications

References 55 publications

Chikungunya Virus Envelope-Specific Human Monoclonal Antibodies with Broad Neutralization Potency

Chikungunya Virus Envelope-Specific Human Monoclonal Antibodies with Broad Neutralization Potency

Epitope Specificity Determines Pathogenicity and Detectability of Anti–Platelet‐Derived Growth Factor Receptor α Autoantibodies in Systemic Sclerosis

Isolation of Human Monoclonal Antibodies That Potently Neutralize Human Cytomegalovirus Infection by Targeting Different Epitopes on the gH/gL/UL128-131A Complex

Contact Info

Product

Resources

About