*7.2.2 Whole-proteome microarrays*

Proteome microarray is a novel alternative to gene expression profiling by microarrays for studying *Chlamydia*–host interaction. Proteins expressed on microarrays display antigenic epitopes, thereby providing an efficient method for immunoprofiling patients and allowing *de novo* identification of disease-related serum antibodies. The technology takes advantage of the recent construction of a whole-proteome microarray using on-chip protein expression of the *C. trachomatis* 895 proteins [106]. Comparison of antibody reactivity patterns allowed the identification of new antigens recognized by known *C. trachomatis* seropositive samples and antigens reacting only with samples from cervical cancer patients [106]. More recently, the whole *C. trachomatis* screening identified antibody patterns associated with pelvic inflammatory disease (PID), tubal factor infertility, chronic pelvic pain (CPP), and ectopic pregnancy that results from a Chlamydial persistent infection [106, 107]. Although protein microarrays have been used in the field of clinical diagnosis for *de novo* identification of antibodies associated with general infection and disease-related serum antibodies, the technique can easily be adapted to the identification of antigen biomarkers of *Chlamydia* persistence.

#### **7.3 In vitro cell systems**

The 2D *in vitro* cell-culture models have been the most widely used models for studying the dynamics of Chlamydial persistence, including its virulence factors and molecular and cellular pathways. The findings of altered morphological forms of *C. psittaci* in infected mouse fibroblasts (L cells) constituted the first *in vitro* model of *Chlamydial* persistence [108]. Since then, the induction of persistent *C. trachomatis* has been studied extensively using different *in vitro* cell lines (Reviewed in Ref. [3]). The different *in vitro* persistence systems have revealed altered *Chlamydial* growth characteristics, for example, enlarged pleomorphic inclusions with a loss of infectivity and cell division. These changes are generally reversible upon removal of the growth inhibitory factor. One advantage of these systems is that they can be used under highly controlled experimental conditions; however, they fail to mimic the complex and dynamically changing structure of *in vivo* human host tissues.

Three-dimensional (3D) cell-culture models based on primary cells are acquiring great importance as a new and robust platform for studying complex biological processes and might be a promising alternative in *C. trachomatis* pathogenetic studies (Reviewed in [109]). The 3D "organoid" models mimic the microenvironment that *C. trachomatis* encounters in the host tissue, allowing a deeper understanding of host–pathogen interactions by promoting direct cell-to-cell contact, interacting with cells of the extracellular matrix and allowing *in vivo* exchange of soluble factors. In addition, 3D cell culture models retain the cellular structural integrity resembling the *in vivo* parental tissue than the 2D cell culture models.

The recent development of Female Reproductive Tract (FRT) Organoid technology is opening up new possibilities to investigate the mechanisms of *Chlamydia* disease in the FRT [Reviewed in 113]. Human and mouse-derived primary cervical epithelial three-dimensional (3D) organoids resembling the *in vivo* FTR native tissue architecture offer a unique possibility to elucidate the dynamics and impact of different infections and co-infections in pathogenesis and carcinogenesis [110]. One advantage of using FTR organoids is that they can be propagated and expanded long term under their optimal culture conditions (≥6 months), thus providing the ideal model to study persistence in *Chlamydia*. For instance, in a human ectocervical organoid model, co-infection with Human papillomavirus (HPV)16 E6E7 slowed down the *C. trachomatis* developmental life cycle by inhibiting the redifferentiation of RBs into EBs, thus inducing persistence [111].
