Adaptive Features of In-Utero Natural Killer Cell Responses to Congenital Cytomegalovirus Infection

ABSTRACT Congenital cytomegalovirus (cCMV) infection is the most common congenital viral infection in the U.S., affecting approximately 18,500 liveborn infants annually. Children with symptomatic or asymptomatic infection at birth can suffer from permanent neurodevelopmental disabilities including cerebral palsy, intellectual impairments, visual deficits, and sensorineural hearing loss. Treatment with the antiviral drug, valganciclovir, improves neurologic outcomes, but drug adverse effects preclude universal antiviral treatment for all cCMV infected infants. Among adults with primary CMV infection, natural killer (NK) cells serve as the first antiviral defense and are rapidly followed by T cell responses that effectively control viral replication. In contrast, cCMV infected infants lack robust antiviral T cell responses to cCMV infection over the first year after birth. These T cells express an exhausted phenotype and fail to control viral replication for months to years. In-utero NK cell responses arise during cCMV infection, but the contribution of these cells to viral control and neurologic outcomes are unstudied. We compared the NK cell immunophenotypes of cCMV infected and uninfected neonates by CITE-seq and found 4 clusters of cytotoxic NK cells induced by cCMV infection. These clusters expressed both activating and inhibitory receptors associated with both NK cells and T cells, and one cluster expressed an inhibitory signature typically observed during T cell exhaustion. Flow cytometry confirmed that cCMV-induced NK cells expressed inhibitory receptors, but NK cells of CMV seropositive adults did not. We therefore hypothesize that in-utero CMV infection induces novel cytotoxic NK cell responses with adaptive-like features that differ from adult responses. We further propose that cytotoxic NK cell function differs between cCMV infected individuals, is altered by antiviral treatment, and may correlate with neurologic outcome. In this project we will characterize the adaptive-like features of cCMV- induced NK cells using blood samples collected longitudinally from a large cohort of cCMV infected infants, CMV uninfected infants, and CMV seropositive adults. Aim 1 will define the immunophenotype and cytotoxic potential of NK cells induced by cCMV infection using CITE-seq, spectral flow cytometry, and in-vitro cytotoxicity assays. Aim 2 will determine the effect of antiviral treatment upon cytotoxic NK cell quantities and immunophenotypes and explore potential associations between NK cell parameters and cCMV neurologic outcomes. Together, these studies will elucidate novel features of NK cell responses induced by in-utero viral infection that differ from adult responses and investigate their relationship to clinical treatments and outcomes among children with congenital CMV infection.