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Vaccination against influenza virus is one of the most efficacious ways to control the spreading of this important disease that is responsible of millions of deaths every year worldwide. However, despite advances in the design of the influenza vaccine, a yearly vaccination is needed to prevent the infection caused by new strains of the virus. The traditional way of evaluating the efficacy of a vaccine is to measure the specific antibody titters generated against the pathogen that follows the administration of the vaccine. However, how the protective antibody response is generated is not fully understood.

One of the most intriguing aspects is this process is the role that innate immunity has in the presentation and the initiation of the protective response. Therefore, the major objective of this project was to study the immunological events that occur in the secondary lymphoid organs right after administration of the vaccine that leads to the production of a protective response. To study this response we have used state-of-the-art imaging techniques, such as 2-photon intravital microscopy, to evaluate “in vivo” the inflammatory reaction and the cell-to-cell interaction. Additionally, we have used other imaging methods such as confocal and traditional molecular and serological techniques to evaluate the effect of the vaccine in the innate compartment.

The first objective of this proposal was related with the characterization of the inflammatory response that followed the administration of an inactivated influenza vaccine in the lymph node (LN). We found that LN macrophages (LNM) play an important role as initiating the inflammatory response and producing pro-inflammatory cytokines right after vaccine administration. However, LNM undergo a necrosis-like death that is reliant on soon after the vaccine arrives to the draining LN (Figure 1).

The second objective of the proposal was related with the study of the effects of the inflammatory response in the innate immune response. We observed that the previously mentioned inflammatory mechanism initiated by LNM promotes lymph node-resident dendritic cell (LNDC) relocation and affects the capacity of the CD11b+ LNDC subtype to capture virus, and express co-stimulatory molecules. Additionally, experimental blocking of the IL-1alpha-induced inflammatory cascade reduced B cell responses, while co-administration of recombinant IL-1alpha increased the humoral response. The stimulation of the IL-1alpha inflammatory pathway might represent a strategy to enhance the antigen-presenting cell function of LNDC and to improve the humoral response against influenza vaccines.

The third objective of the project was related with the study of the mechanism of action of the antibodies generated against influenza virus post vaccination (pv). Using two antibodies, directed towards different parts of the influenza hemagglutinin (HA) molecule, we observed that passive immunization with the previously mentioned antibodies resulted in different degrees of suppression of the antibody responses to the UV-inactivated influenza vaccine, a complete neutralization of the virus reduces the local antibody responses compared to partial neutralization. Remarkably, the inhibition of the humoral response coincided with a reduction in the initial inflammatory reaction measured in the draining lymph node (LN) at 12 h pv, which coincided with a lower activation of the LNDC.

In summary, in this project we study the link between LNM death in response to vaccination and the promotion of DC function in the LN. The stimulation of the IL-1alpha inflammatory pathway might represent a strategy to enhance the antigen-presenting cell function of LNDC and to improve the humoral response against influenza vaccines, especially in the cases where the efficiency of the protection achieved by the vaccine is poor such as in the elderly and immunocompromised people. This is significant for the vaccine industry and can directly help address considerations beneficial particularly for low vaccine responders.

Figure 1: A) Schematic representation of the animal model used in this study. The inactivated influenza vaccine is injected in the footpad of the mice and the immune response is evaluated in the popliteal lymph node (LN). B) The vaccine arrives to the lymph node via the lymphatic system and is captures by lymph node macrophages, which are in charge of the recruitment of LN dendritic cells to this area. C) The inflammatory response is initiated by the death of the LN macrophages and the release of the inflammatory molecules IL-1alpha and IFN-beta which in turn, are involved in the recruitment of dendritic cells (DC). DC are involved in the presentation of viral antigen to T cells (CD4 Type) and the production of specific influenza antibodies by B cells.

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