Detecting the antigen-specific B-cell-response to rabies vaccination.

Rabies is a fatal disease that is still present globally, accounting for 59000
annual deaths. It is primarily transmitted via canine bite incidents, although
other animals (such as bats) can be responsible for transmitting rabies as
well. Adequate pre- and post-exposure treatments exist and have played a major
role in decreasing the death rate of rabies. However, these treatments are not
(financially) accessible universally. The cost of rabies vaccination can be
lowered by decreased dosages per vaccination, or a shorter vaccination regimen.
Current research is carried out to determine whether shorter, lower-dose
vaccination schedules are equally effective to the current WHO-recommended
schedules.
Effectivity of a vaccine/vaccination schedule is usually assessed through RVNA
serology. A titer >0.5 IU/ml is considered protective against rabies. However,
titers will drop eventually, and booster vaccinations need to be reapplied if
titers have dropped below 0.5 IU/ml. To predict long-term protection, other
parameters than serology might contain useful information. Some of these
parameters can be assessed through cellular assays, such as flow cytometric
assays. The quantity, velocity and diversity of the immune response might
predict lasting memory. To assess whether this is the case, an antigen-specific
assay against rabies vaccine needs to be developed.
More and more modern techniques have become available to detect immune cells.
For other diseases, such as dengue or HPV, an antigen-specific cellular assay
is already available. This is not yet the case for rabies. With such an assay,
the composition of the response of B-memory-cells and plasma cells and the
kinetics of the primary and secondary response can be described. Never before
have they been described in such detail and antigen-specifically, and therefore
they might contain useful, still unknown information for the field of
vaccinology and immunology.
Rabies vaccine is the perfect agent for describing vaccination responses. As a
neo-antigen it can be used to assess both the primary and secondary response,
whereas the primary response is harder to assess with other vaccines.
Furthermore, the vaccine contains only virus particles and no adjuvants. The
antigen-specific reaction will therefore always be targeted towards the virus
particles and not to an additive.
This use of this assay is not limited to this. It can also be used to describe
the antigen-specific primary and secondary immune response. These results can
be used as reference values for diagnosing certain immune deficiencies. It can
also be used in monitoring vaccination responses among immunocompromised
patients, and could also provide a faster alternative to serological protection
testing.
The possibilities are vast. Therefore, we would like to develop an assay that
can detect antigen-specific B-cells to rabies vaccination.

The goal of this study is to develop a flow-cytometric assay that is able
detect antigen-specific B-cells to rabies vaccine.

This study consists of two phases, of which the first has been finished in
2022. The current study phase is phase II.
Phase I:
In a single cross-sectional visit of the researchers to the Wageningen
Bioveterinary Research Institute in Lelystad and the LUMC in Leiden, blood will
be drawn from 10 volunteers, 53,5 ml per person (including a 3,5 ml serum
tube), after they have provided informed consent. The volunteers will fill in a
short questionnaire on their rabies vaccination history as well.These
volunteers have received multiple rabies vaccinations in the past, and will NOT
be revaccinated for this study. This blood will be processed and frozen for
future use.
The fresh and stored blood samples will be used to develop, validate and
optimize the assay for flow-cytometric detection of antigen-specific B-cells
against rabies vaccine. The acquired material (blood) is needed solely to
develop and validate the assay. We expect that detection of rabies-specific
B-cells will be possible in these hyperimmunized individuals due to their
repeated previous exposure to (inactivated) rabies and gradual buildup of
immunological memory. Rabies-naïve healthy controls will not have this memory
as they have never been exposed: therefore we expect to see no rabies-specific
B-cells in their blood. Control blood of people who have not been vaccinated
against rabies will be collected from the employees of the departments of IHB,
Infectious Diseases and Medical Microbiology of the LUMC. If they have been
vaccinated against rabies at least three times, they are eligible to
participate as part of the hyperimmunized group. Control blood can also be
acquired from Leiden University students who have been recruited via a general
social media message.
For each blood sample, we will analyze leukocyte differential count and isolate
peripheral blood mononuclear cells. For flow cytometric studies, we will
incubate cells with rabies vaccine and detect vaccine-binding B-cells using
CD19- and CD20-antibodies and an antibody against viral rabies proteins. We
will perform multiple tests to find the optimal vaccine concentration and
antibody clone and concentration. To test these parameters in different
combinations, we need many cells, and therefore sufficient amounts of
leukocytes from hyperimmunized donors will have to be collected. Once an
optimal combination is found, we will validate this flow-cytometric assay among
the blood of multiple hyperimmunized participants and blood of multiple
rabies-naïve donors.

Phase II:
Participants are recruited via email (sent via the respective secretariats of
the departments), posters or social media. They apply for one of the three
groups(group 1: unvaccinated, will receive a single vaccination during study.
group 2: unvaccinated, will not be vaccinated during study. group 3: previously
vaccinated, will receive a single vaccination during study) via email to
ASPERA@lumc.nl, from which they will receive their patient information forms.
Study visits will be scheduled at the vaccination clinic in the LUMC and will
take place on day 0, 7 and 28. After acquiring informed consent from the
participant, the participant will be asked to complete a short questionnaire
containing questions about general health and information on their (rabies)
vaccination history. Only the participants who fulfill the inclusion criteria
will be asked to participate. The information on rabies vaccination history,
including the number of vaccinations, antibody titers, and the corresponding
time frames, will help us interpret the findings. Each visit, peripheral blood
samples (53.5 ml) will be collected and processed and cells and serum will be
stored for future use. The fresh and stored blood samples will be used to
develop, validate and optimize the assay for flow-cytometric/ELISPOT detection
of antigen-specific B-cells against rabies vaccine. The two groups that receive
one rabies vaccination, will receive this vaccination at their first visit.

Recruitment of the previously immunized group will consist of reapproaching
participants of the PREPARE study (NL60550.056.17). They have recently been
vaccinated in the light of this previous study and are therefore reasonably
comparable. If insufficient participants can be recruited from this pool, a
more general approach will be used to recruit participants for this study group.

Should technological developments allow antigen-specific B-cell detection, an
additional study visit might be planned after the first visit for which
participants can be reapproached. This is however completely dependent on the
success of antigen-specific B-cell detection.

Phase II: single intramuscular vaccination with Rabipur vaccine.

The burden of the study is limited and related to venipuncture and vaccination
with a registered rabies vaccine. Risks include mild local or mild reversible
systemic reactions.