Immunology of Ebola Vaccine

Ebolaviruses (EBOV), cause Ebola Virus Disease, a condition characterized primarily by hemorrhagic fevers with remarkably elevated mortality rates. The genus Ebolavirus encompasses five distinct viral species, namely Bundibugyo virus (BDBV), Zaire Ebola virus (ZEBOV), Reston virus (RESTV), Sudan virus (SUDV), and Taï Forest virus (TAFV).

More than 20 human outbreaks have been reported world-wide since the identification of EBOV in the late 1970s. The most common geographical region affected by EBOV outbreaks is Central Africa, with two of the most notable outbreaks occurring in Kikwit, Democratic Republic of Congo in 1995, and Gulu, Uganda in 2000. Outside of Africa, EBOV infections have been reported in countries like Philippines, Italy, United Kingdom, United States of America, and others. Of particular significance is the 2014-2016 West African ZEBOV outbreak, which is known as the most extensively recorded outbreak to date with over 28,000 reported infections and a 40% approximated mortality rate. The outbreak significantly surpassed all preceding ZEBOV outbreaks in terms of geographical coverage, number of impacted individuals, and its disruptive influence on conventional societal activities.

Fatal ZEBOV infection is characterized by flu-like symptoms and high fever followed by multi-organ failure. While case-fatality rates vary between outbreaks and among the Ebola viruses, ZEBOV has been associated with up to 90% lethality. While specific treatment strategies including convalescent plasma, monoclonal antibodies, and/or direct- acting antiviral agents are being pursued, it is unlikely that treatment directed at the individual will be sufficient to control outbreaks. Hence, it is important to investigate prophylactic vaccines that confer protection against Ebola viruses in at-risk populations to prevent future outbreaks. Understanding the durability of these vaccines is of paramount importance.

The recombinant vesicular stomatitis virus-based Ebola vaccine (rVSVΔG-ZEBOV-GP) was approved in 2019 as a single dose in the prevention against Ebola virus disease (EVD). Protection is primarily conferred by antibodies targeting the ZEBOV glycoprotein (GP), and ZEBOV-GP Immunoglobulin G (IgG) memory B cells (MBCs) can be detected in the blood 6 months following vaccination.

It is not fully understood, however, whether a single dose of rVSVΔG-ZEBOV-GP effectively generates germinal center (GC) responses that result in durable immunological memory.

The immune responses that ensue following vaccination consist of a series of highly orchestrated events in GCs of secondary lymphoid organs. The nature of these interactions ultimately dictates the quality and longevity of the immune response generated following vaccination. Long-lived bone marrow plasma cells (BMPCs) and MBCs are the end products of the GC reaction. Previous studies of human B cell immune responses to rVSVΔG-ZEBOV-GP vaccination have focused on the blood compartment. This strategy ignores the critical compartments of draining lymph nodes (dLNs), where GCs are formed, and bone marrow, the major reservoir of BMPCs.

Deuterium labeled water (D2O) or heavy water is chemically nearly the same as normal water (H2O) but the hydrogen atoms are of the heavy isotope deuterium (2H or D), in which the nucleus of the hydrogen atom contains a neutron in addition to the proton. Deuterium is not a radioactive isotope of hydrogen. When a person drinks D2O, it mixes with the body water, which is about 13 gallons in a 180 lb man. Many biological molecules use hydrogen atom from water as "ingredients" and will also use the deuterium from D2O if it is present in the body. Proteins, DNA, RNA, lipids, and other biomolecules become "labeled"; the faster the biomolecules are being synthesized, the more they become labeled with deuterium. The deuterium labeled molecules can be measured by sampling blood and body fluids. After isolating the biomolecules /cells of interest, a sensitive form of mass spectrometry is used to determine the extent of deuterium incorporation. All biomolecules synthesized prior to exposure to heavy water would have hydrogen exclusively (natural levels of deuterium are in trace quantities), while biomolecules synthesized after drinking heavy water will have a mix of hydrogen and deuterium.

This study aims to directly examine the GC response induced in the dLNs after vaccination with rVSVΔG- ZEBOV-GP. The researchers will directly probe ZEBOV-GP-specific GC B cell responses and determine how long these GCs persist after a single vaccine dose. The researchers will determine the frequency of antigen-specific BMPCs that home to bone marrow after vaccination, and whether these BMPCs persist up to 1 year after a single dose. The researchers will determine the frequency of antigen-specific MBCs generated after vaccination. Across all compartments, the researchers will analyze the B cell receptor (BCR) clonal diversity and the degree of somatic hypermutation (SHM) induced by vaccination. This study will determine the degree to which a single dose of rVSVΔG-ZEBOV-GP generates durable GC responses and long lasting humoral immunological memory.

The researchers will use stable isotope labeling to trace antigen-specific B cell responses to vaccination. In one cohort, participants will consume D2O for 14 days post-vaccination, labeling cells proliferating within the first 5-6 weeks. Antigen-specific plasmablasts and early memory B cells, collected at peak frequencies on days 7 and 14, will incorporate high levels of deuterium, measured via gas chromatography-mass spectrometry (GC-MS) analysis of DNA. Memory B cells and bone marrow plasma cells (BMPCs) will then be analyzed at later time points (Days 181 and 366) to assess deuterium dilution by newly divided cells. Another cohort, consuming D2O between days 57-85 post-vaccination, will label cells proliferating during the germinal center response. This approach enables the researchers to evaluate early versus later contributions to long-term memory B cells and antibody-secreting BMPCs.