Chapter 3
Use of Chlamydial Elementary Bodies as Probes to Isolate
Pathogen-Specific Human Monoclonal Antibodies
Christopher L. Pinder, Paul F. McKay, and Robin J. Shattock
Abstract
Chlamydia trachomatis is one of the most prevalent sexually transmitted infectious agents in the world and
the leading cause of infectious blindness. The role of antibodies in the prevention and clearance of infection
is still not fully understood, but the analysis of the immunoglobulin response to novel vaccine candidates is
an important part of many of these studies. In this chapter, we describe a novel method to identify and
isolate Chlamydia-specific memory B cells by fluorescence-activated cell sorting (FACS) using fluorescently
labeled whole bacteria from cryopreserved human PBMC samples. This method allows for live single cells
to be sorted for cell culture, in vitro assays, single-cell RNA sequencing, and cloning of paired heavy and
light chains for recombinant monoclonal antibody production.
Key words Antigen-specific, B cell, Antibody, Chlamydia, Elementary body, Immunoglobulin, IgG
1 Introduction
Chlamydia trachomatis is a Gram-negative intracellular bacterial
pathogen, responsible for over 100 million sexually transmitted
infections a year [1] and nearly 40 million active cases of trachoma,
an ocular chlamydial infection and the leading cause of infectious
blindness [2]. While protective immunity can be developed in the
host by infection, damage caused by the inflammatory response to
the bacteria can cause scarring in infected tissues, resulting in
infertility or blindness [3].
There has therefore been substantial effort to develop a vaccine
to C. trachomatis that would prevent infection and clear the bacteria before damage can occur. Initial work focused on live or attenuated whole-organism vaccination, and while capable of inducing
a protective immune response, this protection was often short-lived
and highly serovar-specific [4, 5]. More recent vaccine designs have
used purified or engineered protein antigens, primarily the immunodominant major outer membrane protein (MOMP), which
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021
19
Use of Chlamydial Elementary Bodies as Probes to Isolate
Pathogen-Specific Human Monoclonal Antibodies
Christopher L. Pinder, Paul F. McKay, and Robin J. Shattock
Abstract
Chlamydia trachomatis is one of the most prevalent sexually transmitted infectious agents in the world and
the leading cause of infectious blindness. The role of antibodies in the prevention and clearance of infection
is still not fully understood, but the analysis of the immunoglobulin response to novel vaccine candidates is
an important part of many of these studies. In this chapter, we describe a novel method to identify and
isolate Chlamydia-specific memory B cells by fluorescence-activated cell sorting (FACS) using fluorescently
labeled whole bacteria from cryopreserved human PBMC samples. This method allows for live single cells
to be sorted for cell culture, in vitro assays, single-cell RNA sequencing, and cloning of paired heavy and
light chains for recombinant monoclonal antibody production.
Key words Antigen-specific, B cell, Antibody, Chlamydia, Elementary body, Immunoglobulin, IgG
1 Introduction
Chlamydia trachomatis is a Gram-negative intracellular bacterial
pathogen, responsible for over 100 million sexually transmitted
infections a year [1] and nearly 40 million active cases of trachoma,
an ocular chlamydial infection and the leading cause of infectious
blindness [2]. While protective immunity can be developed in the
host by infection, damage caused by the inflammatory response to
the bacteria can cause scarring in infected tissues, resulting in
infertility or blindness [3].
There has therefore been substantial effort to develop a vaccine
to C. trachomatis that would prevent infection and clear the bacteria before damage can occur. Initial work focused on live or attenuated whole-organism vaccination, and while capable of inducing
a protective immune response, this protection was often short-lived
and highly serovar-specific [4, 5]. More recent vaccine designs have
used purified or engineered protein antigens, primarily the immunodominant major outer membrane protein (MOMP), which
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021
19