2. Serum or plasma transfers from patients who have survived an infection (convalescent plasma or serum) were a promising therapy prior to development of specific drugs. Currently, these transfers are still used, particularly for emerging infections or deadly infections without a therapy. When serum transfers are effective treatments, monoclonal antibodies may also provide protection. One possible hindrance to these therapies is the potential for antibody dependent enhancement (ADE) of infection.
a. (5 pts) There are two main mechanisms for ADE. Based on your knowledge of antibody functions, what are these two mechanisms?
The antibody dependant enhancement (ADE) is a phenomenon that is responsible for giving rise to severe immunopathogenesis of an infection upon a secondary infection. The ADE aids in the onset of a adverse effects of an otherwise neutral or less severe infection, in creation of a series of events that will conclude production of an serious and severe infection. The ADE is observed usually happens in diseases like dengue or AIDS. In context with the dengue fever once a person a gets infected with the dengue virus the viral load and the diseases pathogenesis decide the severity of the infection leading to Dengue haemorrhagic fever or Dengue shock syndrome. The ADE also plays a major role in this process in the capacity of immunopathogenesis via alteration of certain innate and adaptive immunity pathways. Dengue virus has 4 serotypes and when a person gets infected with one serotype of dengue virus, during and post infection the adaptive immune system produces antibodies against that particular serotype but also produces heterotypic antibodies against other serotypes. The immune protection against the infected serotype will be long lasting while the heterotypic antibodies provide protection only for a limited time (1 -2 years). When this individual gets infected with a different type of a dengue viral serotype the heterotypic antibodies cannot elicit an immune response but hence aids in the disease development and enhances its effects thus increasing the severity. The ADE performs this function in multiple ways but there are two main mechanisms to be followed and those are the intrinsic mechanism and extrinsic mechanism.
In the extrinsic mechanism the heterotypic antibody binding to the virus of another serotype helps the virus to gain entry inside the Dendritic cells (DC) in the skin. The antibody attachment to the virus is suppose to neutralize the viral action but in this scenario instead of eliciting immune action of prohibiting the virus action this binding helps the virus to gain entry inside the body. The binding mainly occur through the Fc receptor and the virus can camouflage itself inside the cell with the help of this process. T
he binding is meant to neutralize the virus surface protein from attaching to the cell, but the
antibody bound to virus also binds to the receptor of the cell, the Fc-region antibody receptor FcγR. This
action brings the virus into close proximity to the virus-specific receptor, and the cell internalizes the
virus through the normal infection route. Once inside the cell they can replicate and produce multiple
viral particles thereby increasing the viral load thus increasing the severity of the infection.
In the intrinsic mechanism the attachment of a different serotype to the virus surface protein can
demodulate and modify the innate immune responses by gaining entry to the DC cells through antibody
mediated route as described above. The alteration occurs along both TLR (toll like receptor) dependant
and independent pathways. The virus can target the TLR dependant pathway by up regulating the
b. (5 pts) How would a researcher determine whether an antibody causes ADE in vitro? Briefly , describe the assay/experiment.
The in vitro determination of whether an antibody can cause ADE can be described as follows. A human cell line such as THP-1 can be used and they can be cultured by using a culture media which contains a serum such a fetal bovine, glutamine and other nutrients and vitamins with correct pH in a CO2 incubator with rotating flask under correct CO2 and O2 percentages. After successful culturing we can perform cell passaging to obtain a functioning monolayer culture of THP-1 cells to perform our experiment. The cell line firstly in being infected by dengue virus (DENV) of serotype 1 (DENV-1) and it is allowed for some time for the infection to spread and register itself in the cell line. After the allocated time a microscopic slide preparation will be performed and will be observed under microscope to detect infection and to confirm infection ELISA assay for DENV-1 will be performed and this will confirm the presence of the DENV-1 antigen. The viral titre calculation will be done via using the flow cytometry by using FACS (fluorescent activated cell sorting) using negative controls with no virus. After allowing sometime for the cell line to alter and modulate itself to the infection, a secondary infection will be given to the cell line of DENV-2 serotype. Same amount of time as previously will be allocated for the infection to successfully register in the cells. The confirmation of the infection will be done by preparing a microscopic slide and observing under the microscope and also by using the ELISA assay for DENV-2 to confirm infection. The viral titer calculation will be done as same as previously by using FACS procedure using negative controls. The viral titer count for DENV-1 will be compared with the viral titer count for the DENV-2 infection and if the ADE phenomenon or antibody causes ADE in vitro the viral titer count will be higher for DENV-2, secondary infection than DENV-1, primary infection. The primary infection and secondary infection both will be performed under same conditions and the conditions for the cell line will be maintained same throughout the experiment.
negative TLR expression regulators and ADE has the ability to shift the TLR signaling into non NF-κB to
signalling along a non-NF-κB pathway. This helps in facilitating the increased replication and viral protein
production inside the cell. ADE can also modify the IFN pathways and they work by reduction of IFN
activity especially IFNβ expression.