My research interest has primarily focused on the physiology of the immune response to viral infection using the mouse cytomegalovirus (MCMV) as a model. My group was first to discover the physiological plasticity of the T cell response to viral infection. Previous studies suggested that CD8 T cells are indispensable for virus control and establishment of latency; on the other hand, we have shown that animals lacking CD8 T cells could still terminate productive infection and establish latency through compensatory responses mediated by CD4 T cells (J Virol. 1990).
Next we have shown that CD4 T cells, although inefficient in virus control after adoptive transfer, play an essential role in terminating productive infection and establishing latency (J Exp Med. 1989). The discovered phenomenon is IFN-γ dependent, since mice lacking either CD4 T cells or IFN-γ failed to control the infection in salivary glands, resulting in lifelong persistency (J Exp Med. 1989, J Virol. 1992). In addition, we were the first to show that antiviral antibodies are not required for the resolution of primary CMV infection and the establishment of latency, but play a key role in limiting recurrent infection (J Exp Med. 1994).
Further, we provided evidence that CD8 and CD4 T cells, IFN-γ as well as NK cells contribute in a hierarchical and redundant fashion to immunosurveillance of CMV latency (J Exp Med. 1998). We also made significant contribution in understanding the role of NK cells in control of CMV infection and regulation of CD8 T cell response (J Virol. 2004, J Virol. 2012). Additionally, we found that inflammatory monocytes and NK cells play a crucial role in DNAM-1-dependent control of CMV infection (J Exp Med. 2016).
Over the past 30 years working in the field of virus immunology at the Department of Histology and Embryology and later at the Center for Proteomics, many of my PhD students have become prosperous scientists who are today principal investigators on highly competitive national and international projects and played a role in raising new generations of immunologists and virologist. Below is a list of the primary research areas currently being conducted at the Center for Proteomics, along with the main investigators leading these projects.
Cytomegaloviruses (CMVs) are large DNA viruses, which utilize a significant portion of their genome to evade the immune response. With a prevalence of > 90% in many mammalian species, they have been an important driving force in the evolution of their hosts’ immune response. This is well demonstrated during MCMV infection of mice (Curr Opin Virol. 2015) where we and others have characterized multiple viral immune evasion proteins that affect nearly every part of our immune system (Figure 1).
Among the various evasive strategies employed by CMVs and many other viruses, modulation of MHC-I is one of the most crucial. Downregulating MHC-I from the surface of an infected cell can protect it from cytotoxic T cells. However, it also makes the cell vulnerable to NK-cell-mediated “missing-self” attack. To evade “missing-self” recognition, two MCMV immunoevasins, namely m04 and MATp1, rescue certain MHC-I alleles from retention or degradation during infection, thus enabling them to reach the cell surface (J Exp Med. 2010, J Exp Med. 2019). We have shown that MATp1/m04-modified altered-self MHC-I molecules engage inhibitory Ly49 NK cell receptors more strongly than MHC-I molecules alone in uninfected cells (J Exp Med. 2010, J Exp Med. 2019). As a result there is an efficient avoidance of NK cell-mediated lysis of infected cells even when MHC-I surface levels are severely diminished. Furthermore, we have also shown how this viral strategy could have prompted the evolution of activating NK cell receptors since at least 3 activating Ly49 receptors specifically recognize MCMV-infected cells via the MATp1/m04/MHC-I complex (J Exp Med. 2009, J Exp Med. 2019) (Figure 2).
In addition, the manipulation of MHC-I molecules by MATp1 seems to affect CD8 T cell responses as well. Preliminary analyses have shown that MATp1 could cause qualitative and quantitative alterations of the presented peptides and thus impair the recognition of infected cells via cytotoxic T lymphocytes. We are currently investigating the effect of the modulation of MHC-I molecules on the short and long-term responses mediated by CD8 T cells. In addition to providing novel insights on the mechanisms by which CMV evade the immune response, this research has the potential to identify new CD8 T cell epitopes that can be used as a tool in various pre-clinical and clinical research.
Main investigators: Vanda Juranić Lisnić, Astrid Krmpotić, Stipan Jonjić
Human cytomegalovirus (HCMV) is a highly prevalent herpesvirus, which can cause severe disease in immunologically immature fetuses and newborns, especially in organ systems that possess a low regenerative capacity. Congenital HCMV (cCMV) infection is a serious medical problem and the most common intrauterine viral infection, with a prevalence of 5 to 10 cases per 1000 live births. Among congenitally infected infants with clinically apparent symptoms (symptomatic infection), almost one-third of them will have CNS sequelae, including microcephaly with significant structural brain damage, retinitis, hearing loss, and vestibular dysfunction. Notably, even infants with asymptomatic cCMV infection can present long-term CNS manifestations (Cell Mol Immunol. 2015). Since CMVs show strict species specificity, thus limiting the use of HCMV in experimental animals, infection following intraperitoneal administration of MCMV into newborn mice efficiently reiterates many aspects of cCMV infection in the CNS. Similarly to infants with cCMV infection, CMV infection of newborn mice is associated with virus-induced inflammation in the CNS, altered brain development exemplified by a delayed migration of neurons from the external granular layer (EGL) of the cerebellum, cerebellar hypoplasia, and sensorineural hearing loss (J Exp Med. 2008, PLoS Pathog. 2015). While studying the immune mechanisms that mediate the immune response and drive pathological changes in the brain during cCMV infection, our lab has determined that a strong inflammatory response in the brain leads to the activation of resident microglia, astrocytosis, and an influx of innate immune cells, including NK, ILC1, and T cells (Eur J Immunol. 2018, Med Microbiol Immunol. 2019, J Exp Med. 2021).
Moreover, effector functions from both resident cells and infiltrating immune cells can not only resolve acute MCMV infections in the CNS, but also mediate the development of immunopathology during CMV infection of the brain (Figure 3a). In terms of understanding chronic inflammation in the brain, our group has shown that CD4 and CD8 T cells are essential for efficient virus control and termination of productive CMV infection in the brain. After resolution of productive infection, CMV establishes lifelong latency in the brain. T cells are retained in the brain for a lifetime as tissue-resident memory (TRM) cells and are required to control virus reactivation (Figure 3b). Our work is also directed towards understanding the neurofunctional alterations upon cCMV infection, microglia-mediated neuropathology and development of potential therapeutic strategies (Figure 3c).
Main investigators: Ilija Brizić, Stipan Jonjić
Viral infections during pregnancy often cause adverse outcomes and birth defects, while the underlying mechanisms are poorly understood. Among those, CMV infection stands out as the most common intrauterine infection in humans, putatively causing early pregnancy loss. We employ MCMV as a model to study the consequences of virus infection on pregnancy outcome and fertility maintenance. We have recently published one of the first in depth analysis of CMV infection and pathogenesis in the ovaries (Immunity. 2021) (Figure 4).
CMV strongly infected the ovaries, especially the corpora lutea (CL) causing a significant drop in serum progesterone levels that diminished pregnancy success by half during early pregnancy. However, despite strong infection of CL and ovarian stroma the virus was excluded from the follicles. Utilizing a large panel of mouse strains, inhibitors and antibody depletions, we have demonstrated that this follicular resistance to infection is a consequence of multiple, overlapping layers of protection: from abundance of gap-junctions, absence of vasculature, strong type I interferon (IFN) responses to interaction of innate immune cells. Our work provided fundamental insights into the impact of CMV infection on pregnancy loss and mechanisms protecting fertility.
Main investigators: Vanda Juranić Lisnić, Berislav Lisnić
Design principle of CMV vectors expressing NKG2D ligands. The fact that CMV has a large genome, with a number of nonessential genes, enables the construction of viral mutants that express antigens of interest. Together with the potential of CMV to reinfect and disseminate irrespective of prior CMV immunity, it makes this virus a perfect vaccine vector. As mentioned above, CMVs develop many immunoevasion mechanisms to avoid host’s immune control and our group has been studying these mechanisms for years. Based on the data on immunology of recombinant viruses lacking immunoevasion genes, we have designed recombinant CMV-based vaccine vectors. We constructed recombinant MCMV in which RAE-1γ, a ligand of activating immune receptor NKG2D, was inserted into the MCMV genome in place of its viral inhibitor m152 (RAE-1γMCMV) (J Clin Invest. 2010) (Figure 5, upper panel). Although highly attenuated in vivo, RAE-1γMCMV efficiently primed and maintained a potent, long-lasting CD8 T cell response to vectored antigen that was comparable or even better than the one induced by wild type (WT) MCMV (Proc Natl Acad Sci U S A. 2013). Immunization with this vector generates memory CD8 T cells which have distinct, effector-like phenotypical features that grossly differ from memory CD8 T cells primed with the vector lacking a RAE-1γ expression (Front Immunol. 2021).
CMV vectors expressing NKG2D ligands and foreign epitopes are highly protective against pathogens and tumors. We have generated RAE1γMCMV-based vectors expressing foreign epitopes and tested their potential as a viral vaccine vectors. Using a backbone of RAE-1γMCMV we have constructed a recombinant virus RAE-1γMCMVList, where the immunodominant CD8 T cell epitope of L. monocytogenes was introduced in place of the MCMV CD8 T cell epitope (Proc Natl Acad Sci U S A. 2013). Mice were immunized with MCMVList or RAE-1γMCMVList and 2 months later challenged with a L. monocytogenes infection. While all unimmunized mice succumbed to infection by day 4, all mice immunized with RAE-1γMCMVList survived the challenge, demonstrating outstanding protective capacity of RAE-1γMCMVList compared to a vector lacking RAE-1γ expression (Figure 5, lower panel, left). In addition, we have shown a potential of RAE-1γMCMV as CD8 T cell-based vaccine against malignant tumors (Proc Natl Acad Sci U S A. 2013). In the melanoma tumor model, mice were immunized with MCMV vector or RAE-1γMCMV vector expressing the SIINFEKL epitope and later challenged with intravenous inoculation of melanoma tumor cells expressing the same SIINFEKL epitope. Mice immunized with RAE-1γMCMV-SIINFEKL had significantly lower number of metastases than mice immunized with MCMV-SIINFEKL, proving the high protective capacity of the RAE-1γMCMV based vaccine vector (Figure 5, lower panel right).
Viral vectors expressing SARS-CoV-2 S protein. Following the emergence of SARS-CoV-2 and subsequent global pandemic, our latest research efforts are focused on testing potential new routes of immunization with commercially available vaccines, as well as developing new alternative vector vaccines against SARS-CoV-2 based on MCMV. We have shown that intranasal immunization of mice with adenoviral vector ChAdOx1-S is superior compared to intramuscular immunization in the induction of systemic and mucosal anti-S IgA response and lung-resident CD8 TRM (Figure 6, left). Both routes elicit systemic anti-S IgG response and SARS-CoV-2 specific cellular response and provide efficient protection of mice infected with MCMV-S (Eur J Immunol. 2022). Additionally, we have generated recombinant MCMV vector vaccines expressing SARS-CoV-2 S protein and analyzed their immunogenicity. Vaccination of mice with MCMV-S recombinant vector induced SARS-CoV-2 antiviral antibodies with a strong neutralizing capacity in the sera of immunized mice (Figure 6, right).
Main investigators: Astrid Krmpotić, Berislav Lisnić, Stipan Jonjić
A part of our research group is devoted to the development and research of new immunotherapeutics based on monoclonal antibodies, and their testing as an antitumor therapy agent using a humanized mouse model. In cooperation with scientists from the Hebrew University in Jerusalem, and the resulting spin-off company Nectin Therapeutics, we have successfully developed several antibodies directed at immune receptors from the nectin family of proteins (J Immunother Cancer. 2020, PNAS. 2009, Eur J Immunol. 2013). These proteins act as immune checkpoints, so targeting them overcomes inhibitory pathways and enables the antitumor activity of immune cells (Figure 7, Cell Mol Immunol. 2019). Our lead clone, an antibody against the poliovirus receptor (PVR, CD155), NTX1088, has recently started the Phase I clinical trial in locally advanced and metastatic solid tumor patients.
Another antibody, directed at the inhibitory receptor TIGIT, discovered by group of Prof. Mandelboim from the Hebrew University in Jerusalem and our group, is currently being investigated. A number of other antibodies are patent protected (PCT/IL2019/050508; PCT/IL2020/050047) and are being extensively tested in preclinical research, partly by our research group.
In addition to the above, as part of our translational effort, we would like to point out the sale of monoclonal antibodies to cellular and viral proteins, produced according to ISO 9001 (Shop | Online Store – Center of Proteomics – Web Shop (capri.com.hr)). The products are recognized and licensed by biotech companies worldwide (Antibodies, Proteins and ELISA kits | Life Science reagents (biorbyt.com)). In collaboration with the company Jadran Galenski laboratorij, a regional pharmaceutical leader, we are investigating antiviral substances that can be used for intranasal administration. In frame of project funded by European Structural and Investment Funds, in cooperation with the Faculty of Veterinary Medicine in Zagreb, we are developing an innovative rapid test for the diagnosis of subclinical mastitis in dairy cows. In addition, recently, we developed a new method for rapid molecular diagnostics of SARS-CoV-2 (Viruses. 2021).
Main investigators: Paola Kučan Brlić, Tihana Lenac Roviš, Stipan Jonjić