accounts for over 60% of the surface of the elementary body (EB),
the extracellular, infectious form of Chlamydia [6–9].
The immune response to Chlamydia has been equally well
studied, mainly using the related C. muridarum in mouse models.
It has been shown that primary immunity to Chlamydia is reliant
on CD4 T lymphocytes [10, 11], data which is supported by the
observation that CD4-deficient HIV patients are more likely to
develop Chlamydia infection and be affected by long-term sequelae
of infection [12]. The role of antibodies and B lymphocytes in
immunity to Chlamydia is less well defined, although it has been
shown that the presence of specific antibodies at the site of exposure
can prevent infection to the same degree as CD4 T cells [13, 14].
For these reasons, analyzing the antibody response to Chlamydia, either from infection with the bacteria or immunization
with a novel vaccine candidate, is of crucial importance. Immunoglobulin repertoire analysis has been significantly advanced in the
last two decades with new developments in single-cell sorting and
sequencing techniques. It is now possible to analyze the antibody
response on a monoclonal basis, identifying individual antibodies
against a specific immunogen that can be useful for research, diagnostics, or design of new and more immunogenic vaccines (Fig. 1).
Fluorescent antigen probes have been of particular use in
pre-screening and enriching for antigen-specific B cells during the
cell sorting process [15–20].
This chapter will outline the use of whole fluorescently labeled
elementary bodies to isolate human memory B cells specific to
C. trachomatis serovars, derived from exposure to both natural
infection with trachoma and to a novel MOMP-based vaccine
antigen.
2 Materials
2.1 PBMC Isolation
and Cryopreservation
1. Phosphate-buffered saline (PBS).
2. Histopaque-1077 [Sigma-Aldrich].
3. PBS containing 1% bovine serum albumin (PBS/BSA).
4. Pasteur pipettes.
5. Freezing medium: 90% fetal bovine serum (FBS), 10%
dimethyl sulfoxide (DMSO).
6. Cell freezing chamber.
2.2 EB Staining
1. SPG buffer: 200 mM sucrose, 20 mM sodium phosphate,
5 mM glutamic acid.
2. Fixation buffer: 3.7% formaldehyde in sterile purified water.
3. Vybrant™ DiO Cell-Labeling solution [Thermo Fisher
Scientific].
20
Christopher L. Pinder et al.
the extracellular, infectious form of Chlamydia [6–9].
The immune response to Chlamydia has been equally well
studied, mainly using the related C. muridarum in mouse models.
It has been shown that primary immunity to Chlamydia is reliant
on CD4 T lymphocytes [10, 11], data which is supported by the
observation that CD4-deficient HIV patients are more likely to
develop Chlamydia infection and be affected by long-term sequelae
of infection [12]. The role of antibodies and B lymphocytes in
immunity to Chlamydia is less well defined, although it has been
shown that the presence of specific antibodies at the site of exposure
can prevent infection to the same degree as CD4 T cells [13, 14].
For these reasons, analyzing the antibody response to Chlamydia, either from infection with the bacteria or immunization
with a novel vaccine candidate, is of crucial importance. Immunoglobulin repertoire analysis has been significantly advanced in the
last two decades with new developments in single-cell sorting and
sequencing techniques. It is now possible to analyze the antibody
response on a monoclonal basis, identifying individual antibodies
against a specific immunogen that can be useful for research, diagnostics, or design of new and more immunogenic vaccines (Fig. 1).
Fluorescent antigen probes have been of particular use in
pre-screening and enriching for antigen-specific B cells during the
cell sorting process [15–20].
This chapter will outline the use of whole fluorescently labeled
elementary bodies to isolate human memory B cells specific to
C. trachomatis serovars, derived from exposure to both natural
infection with trachoma and to a novel MOMP-based vaccine
antigen.
2 Materials
2.1 PBMC Isolation
and Cryopreservation
1. Phosphate-buffered saline (PBS).
2. Histopaque-1077 [Sigma-Aldrich].
3. PBS containing 1% bovine serum albumin (PBS/BSA).
4. Pasteur pipettes.
5. Freezing medium: 90% fetal bovine serum (FBS), 10%
dimethyl sulfoxide (DMSO).
6. Cell freezing chamber.
2.2 EB Staining
1. SPG buffer: 200 mM sucrose, 20 mM sodium phosphate,
5 mM glutamic acid.
2. Fixation buffer: 3.7% formaldehyde in sterile purified water.
3. Vybrant™ DiO Cell-Labeling solution [Thermo Fisher
Scientific].
20
Christopher L. Pinder et al.