Abstract It remains unclear whether antibody-dependent-enhancement (ADE) of dengue infection merely augments viral attachment and entry through Fcγ receptors or immune complex binding to Fcγ ...receptors triggers an intrinsic signaling cascade that changes the viral permissiveness of the cell. Using human dengue–immune sera and novel human monoclonal antibodies against dengue in combination with virologic and immunologic techniques, we found that ADE infection increased the proportion of infected primary human monocytes modestly from 0.2% ± 0.1% (no Ab) to 1.7% ± 1.6% (with Ab) but the total virus output markedly from 2 ± 2 (× 103 ) FFU to 120 ± 153 (× 103 ) FFU. However, this increased virus production was not associated with a reduced secretion of type I interferon or an elevated secretion of anti-inflammatory cytokine, IL-10. These results demonstrate that the regulation of virus production in ADE infection of primary human monocytes is more complex than previously appreciated.
Background.
Human noroviruses are the leading cause of acute viral gastroenteritis, justifying vaccine development despite a limited understanding of strain immunity. After genogroup I (GI).1 ...norovirus infection and immunization, blockade antibody titers to multiple virus-like particles (VLPs) increase, suggesting that GI cross-protection may occur.
Methods.
Immunoglobulin (Ig)A was purified from sera collected from GI.1-infected participants, and potential neutralization activity was measured using a surrogate neutralization assay based on antibody blockade of ligand binding. Human and mouse monoclonal antibodies (mAbs) were produced to multiple GI VLPs to characterize GI epitopes.
Results.
Immunoglobulin A purified from day 14 post-GI.1 challenge sera blocked binding of GI.1, GI.3, and GI.4 to carbohydrate ligands. In some subjects, purified IgA preferentially blocked binding of other GI VLPs compared with GI.1, supporting observations that the immune response to GI.1 infection may be influenced by pre-exposure history. For other subjects, IgA equivalently blocked multiple GI VLPs. Only strain-specific mAbs recognized blockade epitopes, whereas strain cross-reactive mAbs recognized nonblockade epitopes.
Conclusions.
These studies are the first to describe a functional role for serum IgA in norovirus immunity and the first to characterize human monoclonal antibodies to GI strains, expanding our understanding of norovirus immunobiology.
Dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) are life-threatening complications following infection with one of the four serotypes of dengue virus (DENV). At present, no vaccine or ...antiviral therapies are available against dengue. Here, we characterized a panel of eight human or mouse-human chimeric monoclonal antibodies (MAbs) and their modified variants lacking effector function and dissected the mechanism by which some protect against antibody-enhanced lethal DENV infection. We found that neutralizing modified MAbs that recognize the fusion loop or the A strand epitopes on domains II and III of the envelope protein, respectively, act therapeutically by competing with and/or displacing enhancing antibodies. By analyzing these relationships, we developed a novel in vitro suppression-of-enhancement assay that predicts the ability of modified MAbs to act therapeutically against antibody-enhanced disease in vivo. These studies provide new insight into the biology of DENV pathogenesis and the requirements for antibodies to treat lethal DENV disease.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Human connexins 26 and 30 were expressed either through the bicistronic pIRES-EGFP expression vector or as EYFP-tagged chimeras. When transiently transfected in communication-incompetent HeLa cells, ...hCx26–pIRES transfectants were permeable to dyes up to 622
Da, but were significantly less permeable to 759
Da molecules. Under the same conditions, permeability of hCx26–EYFP fusion products was comparable to that of hCx26–pIRES, but with significant increase in diffusion at 759
Da, possibly as a consequence of having selected large fluorescent junctional plaques. Dye transfer was limited to 457
Da in hCx30–EYFP transfectants. When reconstructed from confocal serial sections, fluorescent plaques formed by hCx26–EYFP and hCx30–EYFP appeared irregular, often with long protrusions or deep invagination. Similar plaques were observed following immunostaining both in cells transfected with hCx26–pIRES and in HeLa cells stably transfected with mouse Cx26. Tissue conductance (Tg
j
) displayed significantly smaller values (28.8
±
1.8
nS) for stably transfected mCx26 than transiently transfected hCx26 (43.5
±
3.3
nS). These differences reflected in distinct functional dependence of normalized junctional conductance (
G
j
) on transjunctional voltage (
V
j
). The half-activation voltage for
G
j
was close to ±95 and ±58
mV in mCx26 and hCx26, respectively. The corresponding parameters for hCx30 transfectants were Tg
j
=45.2±3.5
nS and
V
0=±34
mV. These results highlight unexpected differences between mCx26 and hCx26 in this expression system, reinforce the concept that channel permeability may be related to Cx level expression, and indicate that fusion of hCx30 to GFP colour mutants produces channels that are suitable for permeability and gating studies.
Humans who experience a primary dengue virus (DENV) infection develop antibodies that preferentially neutralize the homologous serotype responsible for infection. Affected individuals also generate ...cross-reactive antibodies against heterologous DENV serotypes, which are non-neutralizing. Dengue cross-reactive, non-neutralizing antibodies can enhance infection of Fc receptor bearing cells and, potentially, exacerbate disease. The actual binding sites of human antibody on the DENV particle are not well defined. We characterized the specificity and neutralization potency of polyclonal serum antibodies and memory B-cell derived monoclonal antibodies (hMAbs) from 2 individuals exposed to primary DENV infections. Most DENV-specific hMAbs were serotype cross-reactive and weakly neutralizing. Moreover, many hMAbs bound to the viral pre-membrane protein and other sites on the virus that were not preserved when the viral envelope protein was produced as a soluble, recombinant antigen (rE protein). Nonetheless, by modifying the screening procedure to detect rare antibodies that bound to rE, we were able to isolate and map human antibodies that strongly neutralized the homologous serotype of DENV. Our MAbs results indicate that, in these two individuals exposed to primary DENV infections, a small fraction of the total antibody response was responsible for virus neutralization.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
