Immunological and Virological Prognostic Markers of Human Cytomegalovirus (HCMV) Congenital Infection in Pregnant Women With Primary HCMV Infection.

Human cytomegalovirus (HCMV) establishes a lifelong relationship with its host: primary infection is followed by a latency phase with intermittent reactivation episodes, despite a robust, long-lasting immune response. Primary and non-primary infection (the latter indicating both reactivation and re-infection) is usually asymptomatic in immunocompetent individuals.

Apart from immunocompromised subjects, HCMV is potentially dangerous during pregnancy: the fetus may become infected and develop symptoms at birth or severe long-term sequelae in about 20% of cases (Stagno et al, JAMA 1986; Dollard et al, Rev Med Virol 2007). HCMV is the most common congenital infection with vertical transmission occurring in about 0.64% pregnancies (Cannon \& Davis, BMC Public Health 2005).

Our studies on the T and B cell response to HCMV suggest that a delayed development of the immune response to HCMV primary infection in pregnant women is associated with virus transmission to fetus (Revello et al., J Infect Dis 2006; Lilleri et al., J Infect Dis 2007; 2008; PLoS ONE 2013; Fornara et al., J Clin Immunol 2011, J Med Virol 2015).

More recently, we observed that women transmitting the virus to the fetus had a higher percentage of "short-term effector" (STE) HCMV-specific CD4+ T cells, while an earlier development of "long-term memory" (LTM) cells was associated with a lower risk of virus transmission to the fetus (Mele et al., PLosOne 2017). LTM are characterized by the expression of the receptor for IL-7 (IL-7R), a cytokine involved in the homeostatic maintenance of memory (and naïve) T cells, whereas STE lack expression of IL-7R and are maintained by antigen stimulation. On the same direction, results of another study showed that the earlier development of memory-like CD4+ T cells specific for the pp65 antigen of HCMV (i.e. T cells able to proliferate in vitro in response to the antigen) as determined by a culture ELISPOT is associated with a lower risk of HCMV transmission (Fornara et al., CID 2017).

The limitation of our study of LTM vs STE resides on the fine but cumbersome technique applied, which was based on sorting of the two T-cell subsets and subsequent 3-weeks expansion of multiple T-cell culture replicates (T-cell library), before testing their specificity for HCMV (Geiger et al., J Exp Med 2009). However, our preliminary results show that LTM and STE HCMV-specific T cells can be detected also by direct ex vivo stimulation of T cells and simultaneous determination of IFNγ production (HCMV-specificity) and IL-7R expression (T-cell phenotype) by flow cytometry.

The aims of the present study is to determine the prognostic performance of the combination of different parameters of HCMV-specific T cell response (CD45RA re-expression, LTM phenotype, IL-2 production and lymphoproliferation), in order to be used in the clinical practice to assess the risk of HCMV transmission to the fetus after maternal primary infection. In addition, we will explore whether the presence of memory-like (i.e. expandable) CD4+ T cells able to proliferate in response to other individual HCMV antigens are associated with the risk of virus transmission to the fetus.

HCMV vaccine is actively sought and several candidates are being proposed (Wang \& Fu, Curr Opin Virol 2014) and the definition of immune parameters associated with protection against non-primary infection and virus transmission is mandatory to analyze the effectiveness of different vaccines being developed.