Study of Soluble HLA-G in Congenital Human Cytomegalovirus Infection

Abstract

Human leukocyte antigen-G (HLA-G) is a nonclassical HLA class I antigen that is expressed during pregnancy contributing to maternal-fetal tolerance. HLA-G can be expressed as membrane-bound and soluble forms. HLA-G expression increases strongly during viral infections such as congenital human cytomegalovirus (HCMV) infections, with functional consequences in immunoregulation. In this work we investigated the expression of soluble (s)HLA-G and beta-2 microglobulin (component of HLA) molecules in correlation with the risk of transmission and severity of congenital HCMV infection. We analyzed 182 blood samples from 130 pregnant women and 52 nonpregnant women and 56 amniotic fluid samples from women experiencing primary HCMV infection. The median levels of sHLA-G in maternal serum of women with primary HCMV infection were higher in comparison with nonprimary and uninfected pregnant women (p < 0.001). AF from HCMV symptomatic fetuses presented higher sHLA-G levels in comparison with infected asymptomatic fetuses (p < 0.001), presence of HLA-G free-heavy chain, and a concentration gradient from amniotic fluid to maternal blood. No significant statistical difference of beta-2 microglobulin median levels was observed between all different groups. Our results suggest the determination of sHLA-G molecules in both maternal blood and amniotic fluid as a promising biomarker of diagnosis of maternal HCMV primary infection and fetal HCMV disease.

Figure 1

Maternal serum samples expression of (a) sHLA-G and (b) b2M molecules in nonpregnant and pregnant women. Pregnant women are classified as uninfected and primarily and nonprimarily HCMV infected. p values obtained by Student's t-test and mean ± standard deviation are reported.

Figure 2

Amniotic fluid samples expression of (a) sHLA-G and (b) b2M molecules according to the fetal/neonatal outcome. Maternal serum samples expression of (c) sHLA-G and (d) b2M molecules according to the fetal/neonatal outcome. p values obtained by Student's t-test and mean ± standard deviation are reported.

Figure 3

(a) sHLA-G concentration gradients according to the fetal/neonatal outcome. (b) sHLA-G indexes (%) according to the fetal/neonatal outcome. (c) Representative Western blot analysis of maternal serum and amniotic fluids samples from HCMV primary infected pregnancy subdivided according to the fetal/neonatal outcome. The analysis were performed after immunoprecipitation with anti-b2M associated HLA-G moAb (MEM-G9, Exbio) (lines 1 to 3) and anti-free HLA-G HC moAb (MEMG-1, Exbio) (lines 4 to 6). JEG3 cell line supernatants were used as positive control (M) and the positivity for HLA-G molecule was evidenced at 39 kD.