Human cytomegalovirus (HCMV) infection remains a major complication for solid organ transplant recipients (SOTRs). The aim of this study was to evaluate the role of HCMV-specific T cell immunity measured at the time of the HCMV-DNA peak in predicting the spontaneous clearance of infection. The performance of cytokine flow cytometry using infected dendritic cells (CFC-iDC), infected cell lysate (CFC-iCL) and pp65 peptide pool (CFC-pp65 pool) as stimuli, as well as ELISPOT assays using infected cell lysate (ELISPOT-iCL) and the pp65 peptide pool (ELISPOT-pp65 pool), was analysed. Among the 40 SOTRs enrolled, 16 patients (40%) required antiviral treatment for an HCMV infection (Non-Controllers), while the others spontaneously cleared the infection (Controllers). At the HCMV-DNA peak, the number of HCMV-specific CD4⁺ T cells detected by the CFC-iDC, CFC-iCL and CFC-pp65 pool assays in Controllers was higher than that detected in Non-Controllers, while no difference was observed in terms of HCMV-specific CD8⁺ T cell response. The same trend was observed when the HCMV-specific T cell response was measured by ELISPOT-iCL and ELISPOT-pp65 pool. We observed that the CD4⁺ CFC-pp65 pool assay was the best predictor of self-resolving HCMV infection at the time of the HCVM-DNA peak. The CFC-pp65 pool assay is able to discriminate between CD4⁺ and CD8⁺ T cell responses and could be used in daily clinical practice.

BACKGROUND. Most humans have been infected with cytomegalovirus (CMV) by midlife without clinical signs of disease. However, in settings in which the immune system is undeveloped or compromised, the virus is not adequately controlled and consequently presents a major infectious cause of both congenital disease during pregnancy as well as opportunistic infection in children and adults. With clear evidence that risk to the fetus varies with gestational age at the time of primary maternal infection, further research on humoral responses to primary CMV infection during pregnancy is needed. METHODS. Here, systems serology tools were applied to characterize antibody responses to CMV infection in pregnant and nonpregnant women experiencing either primary or chronic infection. RESULTS. Whereas strikingly different antibody profiles were observed depending on infection status, limited differences were associated with pregnancy status. Beyond known differences in IgM responses used clinically for identification of primary infection, distinctions observed in IgA and FcγR-binding antibodies and among antigen specificities accurately predicted infection status. Machine learning was used to define the transition from primary to chronic states and predict time since infection with high accuracy. Humoral responses diverged over time in an antigen-specific manner, with IgG3 responses toward tegument decreasing over time as typical of viral infections, while those directed to pentamer and glycoprotein B were lower during acute and greatest during chronic infection. CONCLUSION. In sum, this work provides insights into the antibody response associated with CMV infection status in the context of pregnancy, revealing aspects of humoral immunity that have the potential to improve CMV diagnostics. FUNDING. CYMAF consortium and NIH NIAID.

Background. Pregnant women are more susceptible to severe disease associated with SARS-CoV-2 infection. We performed a prospective study to analyze the inflammatory and immune profile after SARS-CoV-2 infection occurring in vaccinated or non-vaccinated pregnant women and their newborns. Methods. Twenty-five pregnant women with SARS-CoV-2 infection were enrolled, and sixteen cord blood samples were obtained at delivery. Results. We observed that IL-1β, TNF-α, Eotaxin, MIB-1β, VEGF, IL-15, IL-2, IL-5, IL-9, IL-10 and IL-1ra levels were significantly higher in vaccinated than non-vaccinated mothers. Furthermore, the newborns of the vaccinated mothers produced higher levels of IL-7, IL-5 and IL-12 compared to the newborns of non-vaccinated mothers. Anti-Spike (S) IgG levels were significantly higher in all vaccinated mothers and their newborns compared to the non-vaccinated group. We found that 87.5% of vaccinated women and 66.6% of non-vaccinated women mounted an S-specific T-cell response quantified by ELISpot assay. Moreover, 75.0% of vaccinated mothers and 38.4% of non-vaccinated mothers showed S-specific CD4⁺ T-cell proliferative response. The T-helper subset response was restricted to CD4⁺ T_h1 in both vaccinated and non-vaccinated women. Conclusion. A higher level of cytokines, IgG antibodies and memory T cells was noted in the vaccinated women. Furthermore, the maternal IgG antibody trans-placental transfer occurred more frequently in vaccinated mothers and may protect the newborn.

In the setting of infectious diseases, antibodies show different functions beyond neutralizing activity. In this study, we investigated the activation of NK cells in vitro in the presence of human cytomegalovirus (HCMV)-specific antibodies and their potential role in the control of HCMV infection through antibody-dependent cell cytotoxicity (ADCC). Retinal pigmented epithelial cells (ARPE-19) infected with the HCMV strain VR1814 were co-cultured with cytokine-activated peripheral blood mononuclear cells (PBMCs) in the presence of sera collected from 23 HCMV-seropositive and 9 HCMV-seronegative donors. Moreover, 13 pregnant women sampled 3 and 6 months after HCMV primary infection and 13 pregnant women with pre-conception immunity were tested and compared. We determined the percentage of activated NK cells via the analysis of CD107a expression as a marker of degranulation. Significantly higher levels of NK-cell activation were observed using 1/100 and 1/10 dilutions of sera from HCMV-seropositive individuals, and when cells were infected for 96 and 120 h, suggesting that NK cells are activated by antibodies directed against late antigens. In the absence of serum NK cells, activation was negligible. In seropositive subjects, the median percentages of CD107a-positive NK cells in the presence of autologous serum and pooled HCMV-positive serum were similar (14.03% [range 0.00–33.56] and 12.42% [range 1.01–46.00], respectively), while NK-cell activation was negligible using an HCMV-negative serum pool. In HCMV-seronegative subjects, the median percentage of activated NK cells was 0.90% [range 0.00–3.92] with autologous serum and 2.07% [0.00–5.76] in the presence of the HCMV-negative serum pool, while it was 8.97% [0.00–26.49] with the pool of HCMV-positive sera. NK-cell activation using hyperimmune globulin is comparable to what is obtained using autologous serum. Sera from subjects at 3 and 6 months post primary infection showed a lower capacity of NK-cell activation than sera from subjects with past infection (p < 0.001). NK activation against HCMV-infected epithelial cells is dependent on the presence of HCMV-specific antibodies. This serum activity increases with time after the onset of HCMV infection. The protective role of NK-cell activation by HCMV-specific serum antibodies should be verified in clinical settings.

Human Cytomegalovirus (HCMV) transcripts (including UL21.5 mRNA) have been found to be packaged in virions and their detection in plasma may indicate the presence of infectious viral particles. Objective of this study was to verify whether UL21.5 mRNA detected in the plasma was indeed encapsulated in viral particles, representing an indirect marker of active replication. To distinguish between virion-packaged and free-floating RNA, plasma samples from 22 immunocompromised patients were tested before and after ribonuclease (RNAse) digestion. UL21.5 mRNA was detected 1-2 weeks prior to preemptive therapy administration in 20 episodes (from 18 patients) of clinically significant DNAemia, while it was undetectable in three of the four patients with transient, self-resolving DNAemia. After RNAse digestion, UL21.5 mRNA was still detectable, with a median reduction of 0.1 (IQ range 0-0.3) Log ₁₀ . Concentrations of UL21.5 mRNA in plasma correlated significantly with HCMV DNA in whole blood or plasma (R=0.67), and 75% of samples positive for UL21.5 mRNA had HCMV DNA concentrations above 10 ⁴ copies/ml blood or 10 ³ copies/ml plasma. Moreover, UL21.5 mRNA was positive in patients who developed HCMV infection resistant to letermovir or maribavir, whereas it was undetectable in plasma of patients with transient self-resolving DNAemia blips during letermovir prophylaxis (not associated with drug-resistance). HCMV UL21.5 mRNA in plasma is virion-associated and represents a marker for productive HCMV infection. The determination of UL21.5 mRNA could improve current strategies for the management of HCMV infection in immunocompromised patients.