Mijo Golemac

Passive Immunization Reduces Murine Cytomegalovirus-Induced Brain Pathology in Newborn Mice

ABSTRACT Human cytomegalovirus (HCMV) is the most frequent cause of congenital viral infections in humans and frequently leads to long-term central nervous system (CNS) abnormalities that include learning disabilities, microcephaly, and hearing loss. The pathogenesis of the CNS infection has not been fully elucidated and may arise as a result of direct damage of CMV-infected neurons or indirectly secondary to inflammatory response to infection. We used a recently established model of mouse CMV (MCMV) infection in newborn mice to analyze the contribution of humoral immunity to virus clearance from the brain. In brains of MCMV-infected newborn mice treated with immune serum, the titer of infectious virus was reduced below detection limit, whereas in the brains of mice receiving control (nonimmune) serum significant amounts of virus were recovered. Moreover, histopathological and immunohistological analyses revealed significantly less CNS inflammation in mice treated with immune serum. Treatment with MCMV-specific monoclonal antibodies also resulted in the reduction of virus titer in the brain. Recipients of control serum or irrelevant antibodies had more viral foci, marked mononuclear cell infiltrates, and prominent glial nodules in their brains than mice treated with immune serum or MCMV-specific antibodies. In conclusion, our data indicate that virus-specific antibodies have a protective role in the development of CNS pathology in MCMV-infected newborn mice, suggesting that antiviral antibodies may be an important component of protective immunological responses during CMV infection of the developing CNS.

Murine CMV-Induced Hearing Loss Is Associated with Inner Ear Inflammation and Loss of Spiral Ganglia Neurons

Congenital human cytomegalovirus (HCMV) occurs in 0.5–1% of live births and approximately 10% of infected infants develop hearing loss. The mechanism(s) of hearing loss remain unknown. We developed a murine model of CMV induced hearing loss in which murine cytomegalovirus (MCMV) infection of newborn mice leads to hematogenous spread of virus to the inner ear, induction of inflammatory responses, and hearing loss. Characteristics of the hearing loss described in infants with congenital HCMV infection were observed including, delayed onset, progressive hearing loss, and unilateral hearing loss in this model and, these characteristics were viral inoculum dependent. Viral antigens were present in the inner ear as were CD3+ mononuclear cells in the spiral ganglion and stria vascularis. Spiral ganglion neuron density was decreased after infection, thus providing a mechanism for hearing loss. The lack of significant inner ear histopathology and persistence of inflammation in cochlea of mice with hearing loss raised the possibility that inflammation was a major component of the mechanism(s) of hearing loss in MCMV infected mice.

Superior induction and maintenance of protective CD8 T cells in mice infected with mouse cytomegalovirus vector expressing RAE-1γ

Significance There are a number of pathogens for which the immunity acquired postinfection does not fully protect against reinfection and disease. Therefore, vaccines offering superior protection compared with the protection following natural infection are needed. Due to a unique pattern of immune response induced by cytomegaloviruses (CMVs), live attenuated CMVs are attractive candidates for vaccine vectors. Here we have demonstrated that a recombinant CMV vector expressing RAE-1γ, a cellular ligand for activating NKG2D receptor expressed on several types of immune cells, has tremendous potential for subverting viral immunoevasion and enhancing the efficiency of the CD8 T-cell response against vectored antigens. This study demonstrates a significant new approach in designing T-cell–based vaccine vectors. Due to a unique pattern of CD8 T-cell response induced by cytomegaloviruses (CMVs), live attenuated CMVs are attractive candidates for vaccine vectors for a number of clinically relevant infections and tumors. NKG2D is one of the most important activating NK cell receptors that plays a role in costimulation of CD8 T cells. Here we demonstrate that the expression of CD8 T-cell epitope of Listeria monocytogenes by a recombinant mouse CMV (MCMV) expressing the NKG2D ligand retinoic acid early-inducible protein 1-gamma (RAE-1γ) dramatically enhanced the effectiveness and longevity of epitope-specific CD8 T-cell response and conferred protection against a subsequent challenge infection with Listeria monocytogenes. Unexpectedly, the attenuated growth in vivo of the CMV vector expressing RAE-1γ and its capacity to enhance specific CD8 T-cell response were preserved even in mice lacking NKG2D, implying additional immune function for RAE-1γ beyond engagement of NKG2D. Thus, vectors expressing RAE-1γ represent a promising approach in the development of CD8 T-cell–based vaccines.

IL-33/ST2 pathway drives regulatory T cell dependent suppression of liver damage upon cytomegalovirus infection.

Regulatory T (Treg) cells dampen an exaggerated immune response to viral infections in order to avoid immunopathology. Cytomegaloviruses (CMVs) are herpesviruses usually causing asymptomatic infection in immunocompetent hosts and induce strong cellular immunity which provides protection against CMV disease. It remains unclear how these persistent viruses manage to avoid induction of immunopathology not only during the acute infection but also during life-long persistence and virus reactivation. This may be due to numerous viral immunoevasion strategies used to specifically modulate immune responses but also induction of Treg cells by CMV infection. Here we demonstrate that liver Treg cells are strongly induced in mice infected with murine CMV (MCMV). The depletion of Treg cells results in severe hepatitis and liver damage without alterations in the virus load. Moreover, liver Treg cells show a high expression of ST2, a cellular receptor for tissue alarmin IL-33, which is strongly upregulated in the liver of infected mice. We demonstrated that IL-33 signaling is crucial for Treg cell accumulation after MCMV infection and ST2-deficient mice show a more pronounced liver pathology and higher mortality compared to infected control mice. These results illustrate the importance of IL-33 in the suppressive function of liver Treg cells during CMV infection.

Activation of Innate and Adaptive Immunity by a Recombinant Human Cytomegalovirus Strain Expressing an NKG2D Ligand

Development of an effective vaccine against human cytomegalovirus (HCMV) is a need of utmost medical importance. Generally, it is believed that a live attenuated vaccine would best provide protective immunity against this tenacious pathogen. Here, we propose a strategy for an HCMV vaccine that aims at the simultaneous activation of innate and adaptive immune responses. An HCMV strain expressing the host ligand ULBP2 for the NKG2D receptor was found to be susceptible to control by natural killer (NK) cells, and preserved the ability to stimulate HCMV-specific T cells. Infection with the ULBP2-expressing HCMV strain caused diminished cell surface levels of MHC class I molecules. While expression of the NKG2D ligand increased the cytolytic activity of NK cells, NKG2D engagement in CD8+ T cells provided co-stimulation and compensated for lower MHC class I expression. Altogether, our data indicate that triggering of both arms of the immune system is a promising approach applicable to the generation of a live attenuated HCMV vaccine.

The specific NK cell response in concert with perforin prevents CD8+ T cell-mediated immunopathology after mouse cytomegalovirus infection

Abstract Natural killer (NK) and CD8+ T cells play a crucial role in the control of mouse cytomegalovirus (MCMV) infection. These effector cells exert their functions by releasing antiviral cytokines and by cytolytic mechanisms including perforin activation. In addition to their role in virus control, NK cells play an immunoregulatory role since they shape the CD8+ T cell response to MCMV. To investigate the role of perforin-dependent cytolytic mechanism in NK cell modulation of CD8+ T cell response during acute MCMV infection, we have used perforin-deficient C57BL/6 mice (Prf1−/−) and have shown that virus control by CD8+ T cells in Prf1−/− mice is more efficient if NK cells are activated by the engagement of the Ly49H receptor with the m157 MCMV protein. A lack of perforin results in severe liver inflammation after MCMV infection, which is characterized by immunopathological lesions that are more pronounced in Prf1−/− mice infected with virus unable to activate NK cells. This immunopathology is caused by an abundant infiltration of activated CD8+ T cells. The depletion of CD8+ T cells has markedly reduced pathohistological lesions in the liver and improved the survival of Prf1−/− mice in spite of an increased viral load. Altogether, the results of our study suggest that a lack of perforin and absence of the specific activation of NK cells during acute MCMV infection lead to an unleashed CD8+ T cell response that is detrimental for the host.

Brain-resident memory CD8+ T cells induced by congenital CMV infection prevent brain pathology and virus reactivation

Congenital HCMV infection is a leading infectious cause of long‐term neurodevelopmental sequelae. Infection of newborn mice with mouse cytomegalovirus (MCMV) intraperitoneally is a well‐established model of congenital human cytomegalovirus infection, which best recapitulates the hematogenous route of virus spread to brain and subsequent pathology. Here, we used this model to investigate the role, dynamics, and phenotype of CD8+ T cells in the brain following infection of newborn mice. We show that CD8+ T cells infiltrate the brain and form a pool of tissue‐resident memory T cells (TRM cells) that persist for lifetime. Adoptively transferred virus‐specific CD8+ T cells provide protection against primary MCMV infection in newborn mice, reduce brain pathology, and remain in the brain as TRM cells. Brain CD8+ TRM cells were long‐lived, slowly proliferating cells able to respond to local challenge infection. Importantly, brain CD8+ TRM cells controlled latent MCMV and their depletion resulted in virus reactivation and enhanced inflammation in brain.