The Role of Heterologous Immunity in Viral Co-Infections and Neonatal Immunity: A Dissertation

The dynamics of T cell responses have been extensively studied during single virus infection of naïve mice. During a viral infection, viral antigen is presented in the context of MHC class I molecules on the surface of infected cells. Activated CD8 T cells that recognized viral antigens mediate clearance of virus through lysis of these infected cells. We hypothesize that the balance between the replicating speed of the virus and the efficiency at which the T cell response clears the virus is key in determining the disease outcome of the host. Lower T cell efficiency and delayed viral clearance can lead to extensive T cellmediated immunopathology and death in some circumstances. To examine how the efficiency of the immune response would impact immunopathology we studied several viral infection models where T cell responses were predicted to be less than optimal: 1. a model of co-infection with two viruses that contain a crossreactive epitope, 2. a viral infection model where a high dose infection is known to induce clonal exhaustion of the CD8 T cell response, 3. a neonatal virus infection model where the immune system is immature and 4. A model of beneficial heterologous immunity and T cell crossreactivity where mice are immunized as neonates when the T cell pool is still developing. Model 1. Simultaneous co-infections are common and can occur from mosquito bites, contaminated needle sticks, combination vaccines and the simultaneous administration of multiple vaccines. Using two distantly related arenaviruses, lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PICV), we questioned if immunological T cell memory and subsequent protection would be altered following a simultaneous co-infection, where two immune responses are generated within the same host at the same time. Coinfection with these two viruses, which require CD8 T cell responses to clear, resulted in decreased immune protection and enhanced immunopathology after challenge with either virus. After primary co-infection, each virus-specific immune response impacted the other as they competed within the same host and resulted in several significant differences in the CD8 T cell responses compared to mice infected with a single virus. Co-infected mice had a dramatic decrease in the overall size of the LCMV-specific CD8 T cell response and variability in which virus-specific response dominated, along with skewing in the immunodominance hierarchies from the normal responses found in single virus infected mice. The reduction in the number of LCMV-specific CD8 memory T cells, specifically cells with an effector memory-like phenotype, was associated with higher viral loads and increased liver pathology in co-infected mice upon LCMV challenge. The variability in the immunodominance hierarchies of co-infected mice resulted in an enhanced cross-reactive response in some mice that mediated enhanced immune-mediated fat pad pathology during PICV challenge. In both viral challenge models, an ineffective memory T cell response in co-infected mice facilitated increased viral replication, possibly leading to enhanced and prolonged accumulation of secondary effector T cells in the tissues, thereby leading to increased immune pathology. Thus, the magnitude and character of memory CD8 T cell responses in simultaneous co-infections differed substantially from those induced by single immunization. This has implications for the design of combination vaccines and scheduling of simultaneous immunizations. Model 2. The balance between protective immunity and immunopathology often determines the fate of the virus-infected host. Several human viruses have been shown to induce a wide range of severity of disease. Patients with hepatitis B virus (HBV), for example, show disease progression ranging from acute resolving infection to a persistent infection and fulminant hepatitis. Certain rapidly replicating viruses have the ability to clonally exhaust the T cell response, such as HBV and hepatitis C virus (HCV) in humans and the clone 13 strain of LCMV in mice. How rapidly virus is cleared is a function of initial viral load, viral replication rate, and efficiency of antigen-specific T cells. By infecting mice with three different inocula of LCMV clone 13, we questioned how the race between virus replication and T cell responses could result in different disease outcomes. A low dose of LCMV generated efficient CD8 T effector cells, which cleared the virus with minimal lung and liver pathology. A high dose of LCMV resulted in clonal exhaustion of T cell responses, viral persistence and little immunopathology. An intermediate dose only partially exhausted the CD8 T cell responses and was associated with significant mortality, and the surviving mice developed viral persistence and massive immunopathology, including necrosis of the lungs and liver. This was a T cell-mediated disease as T cell-deficient mice had no pathology and became persistently infected like mice infected with a high dose of LCMV clone 13. This suggests that for non-cytopathic viruses like LCMV, HCV and HBV, clonal exhaustion may be a protective mechanism preventing severe immunopathology and death. Model 3. Newborns are more susceptible to infections due to their lack of immunological memory and under-developed immune systems. Passive maternal immunity helps protect neonates until their immune systems have matured. We questioned if a noncytolytic virus that produces strong T cell responses in adult mice would also induce an equally effective response in neonatal mice. Neonates were infected with very low doses of LCMV Armstrong and surprisingly the majority succumbed to infection between days 7-11, which is the peak of the T cell response in adult mice infected with LCMV. Death was caused by T cell-dependent pathology and not viral load as 100% of T cell deficient neonates survived with minimal lung and liver pathology. This is similar to the adult model of medium dose LCMV clone 13, but T cell responses in neonates were not partially clonal exhausted. Furthermore, surviving neonates were not persistently infected, clearing virus by day 14 post infection. In adult mice direct intracranial infection leads to LCMV replication and CD8 T cell infiltration in the central nervous system (CNS), causing CD8 T cell-mediated death. However, this does not occur in adults during LCMV intraperitoneal (ip) infections. We questioned if unlike adults LCMV could be gaining access to the CNS in neonates following ip infection. Replicating LCMV was found in the brain of neonates after day 5 post infection along with virus-specific CD8 T cells producing IFNγ at day 9 post infection. Neonates lacking perforin had complete survival when followed until day 14 post infection, suggesting perforin-mediated T cell-dependent immunopathology within the CNS of neonates was causing death after LCMV infection. Passive immunity from LCMV-immune mothers also protected 100% of pups from death by helping control viral load early in infection. We believe that the maternal antibody compensates for the immature innate immune response of neonates and controls viral replication early so the neonatal T cell response induced less immunopathology. Neonates are commonly thought to have less functional immune systems, but these results show that neonates are capable of producing strong T cell responses that contribute to increased mortality. Model 4. Due to their enhanced susceptibility to infection neonatal and infant humans receive multiple vaccines. Several non-specific effects from immunizations have been observed, for example, measles or Bacillus Calmette- Guerin (BCG) vaccines have been linked to decreased death of children from infections other than measles virus or tuberculosis. These studies mirror the concepts of beneficial heterologous immunity, where previous immunization with an unrelated pathogen can result in faster viral clearance. LCMV-immune mice challenged with vaccinia virus (VV) have lower viral loads then naïve mice and survive lethal infections, but some mice do develop fat pad immunopathology in the form of panniculitis or acute fatty necrosis (AFN). We questioned how immunological T cell memory formed during the immature neonatal period would compare to memory generated in fully mature adults during a heterologous viral challenge. Mice immunized as neonates had comparable reduction in VV load and induction of AFN, indicating that heterologous immunity is established during viral infections early in life. Interestingly, the LCMV-specific memory populations that expanded in mice immunized as neonates differed from that of mice immunized as adults. In adult mice 50% of the mice have an expansion of LCMVNP205- specific CD8 T cells while the majority of neonates expanded the LCMVGP34- specific CD8 T cell pool. This alteration in dominant crossreactivities may be due to the limited T cell receptor repertoire of neonatal mice. In naïve neonatal mice we found altered Vβ repertoires within the whole CD8 T cell pool. Furthermore, there was altered Vβ usage within virus-specific responses compared to adult mice and a wide degree of variability between individual neonates, suggesting enhanced private specificity of the TCR repertoire. Beneficial heterologous immunity is maintained in neonates, but there was altered usage of crossreactive responses. As neonatal mice were found to be so sensitive to LCMV infection we questioned if neonates could control another arena virus that did not replicate as efficiently in mice, PICV. Unlike LCMV infection, neonatal mice survived infection with PICV even with adult-like doses. However, viral clearance was protracted in neonates compared to adults, but was cleared from fat pad and kidney by day 11 post infection. The peak of the CD8 T cell response was similarly delayed. PICV infected neonates showed dose-dependent PICV-specific CD8 T cell responses, which were similar to adult responses by frequency, but not total number. As with LCMV infection there were changes in immunodominance hierarchies in neonates. Examination of the immunodominance hierarchies of PICV-infected neonates showed that there were adult-like responses to the dominant NP38- specific response, but a loss of the NP122-specific response. Six weeks post neonatal infection mice were challenged with LCMV Armstrong and there was a strong skewing of the PICV immunodominance hierarchy to the crossreactive NP205-specific response. These data further support the hypothesis that heterologous immunity and crossreactivity develop following neonatal immunization, much as occurs in adults, although TCR repertoire and crossreactive patterns may differ. Changing the balance between T cell efficiency and viral load was found to altered the severity of the developing immunopathology after viral infection

T cells in the brain enhance neonatal mortality during peripheral LCMV infection

In adult mice the severity of disease from viral infections is determined by the balance between the efficiency of the immune response and the magnitude of viral load. Here, the impact of this dynamic is examined in neonates. Newborns are highly susceptible to infections due to poor innate responses, lower numbers of T cells and Th2-prone immune responses. Eighty-percent of 7-day old mice, immunologically equivalent to human neonates, succumbed to extremely low doses (5 PFU) of the essentially non-lethal lymphocytic choriomeningitis virus (LCMV-Armstrong) given intraperitoneally. This increased lethality was determined to be dependent upon poor early viral control, as well as, T cells and perforin as assessed in knockout mice. By day 3, these neonatal mice had 400-fold higher viral loads as compared to adults receiving a 10,000-fold (5X104 PFU) higher dose of LCMV. The high viral load in combination with the subsequent immunological defect of partial CD8 T cell clonal exhaustion in the periphery led to viral entry and replication in the brain. Within the brain, CD8 T cells were protected from exhaustion, and thus were able to mediate lethal immunopathology. To further delineate the role of early viral control, neonatal mice were infected with Pichinde virus, a less virulent arenavirus, or LCMV was given to pups of LCMV-immune mothers. In both cases, peak viral load was at least 29-fold lower, leading to functional CD8 T cell responses and 100% survival

PC61 (anti-CD25) treatment inhibits influenza A virus-expanded regulatory T cells and severe lung pathology during a subsequent heterologous lymphocytic choriomeningitis virus infection

Prior immunity to influenza A virus (IAV) in mice changes the outcome to a subsequent lymphocytic choriomeningitis virus (LCMV) infection and can result in severe lung pathology, similar to that observed in patients that died of the 1918 H1N1 pandemic. This pathology is induced by IAV-specific memory CD8(+) T cells cross-reactive with LCMV. Here, we discovered that IAV-immune mice have enhanced CD4(+) Foxp3(+) T-regulatory (Treg) cells in their lungs, leading us to question whether a modulation in the normal balance of Treg and effector T-cell responses also contributes to enhancing lung pathology upon LCMV infection of IAV-immune mice. Treg cell and interleukin-10 (IL-10) levels remained elevated in the lungs and mediastinal lymph nodes (mLNs) throughout the acute LCMV response of IAV-immune mice. PC61 treatment, used to decrease Treg cell levels, did not change LCMV titers but resulted in a surprising decrease in lung pathology upon LCMV infection in IAV-immune but not in naive mice. Associated with this decrease in pathology was a retention of Treg in the mLN and an unexpected partial clonal exhaustion of LCMV-specific CD8(+) T-cell responses only in IAV-immune mice. PC61 treatment did not affect cross-reactive memory CD8(+) T-cell proliferation. These results suggest that in the absence of IAV-expanded Treg cells and in the presence of cross-reactive memory, the LCMV-specific response was overstimulated and became partially exhausted, resulting in a decreased effector response. These studies suggest that Treg cells generated during past infections can influence the characteristics of effector T-cell responses and immunopathology during subsequent heterologous infections. Thus, in humans with complex infection histories, PC61 treatment may lead to unexpected results

Increased Immune Response Variability during Simultaneous Viral Coinfection Leads to Unpredictability in CD8 T Cell Immunity and Pathogenesis

T cell memory is usually studied in the context of infection with a single pathogen in naive mice, but how memory develops during a coinfection with two pathogens, as frequently occurs in nature or after vaccination, is far less studied. Here, we questioned how the competition between immune responses to two viruses in the same naive host would influence the development of CD8 T cell memory and subsequent disease outcome upon challenge. Using two different models of coinfection, including the well-studied lymphocytic choriomeningitis (LCMV) and Pichinde (PICV) viruses, several differences were observed within the CD8 T cell responses to either virus. Compared to single-virus infection, coinfection resulted in substantial variation among mice in the size of epitope-specific T cell responses to each virus. Some mice had an overall reduced number of virus-specific cells to either one of the viruses, and other mice developed an immunodominant response to a normally subdominant, cross-reactive epitope (nucleoprotein residues 205 to 212, or NP205). These changes led to decreased protective immunity and enhanced pathology in some mice upon challenge with either of the original coinfecting viruses. In mice with PICV-dominant responses, during a high-dose challenge with LCMV clone 13, increased immunopathology was associated with a reduced number of LCMV-specific effector memory CD8 T cells. In mice with dominant cross-reactive memory responses, during challenge with PICV increased immunopathology was directly associated with these cross-reactive NP205-specific CD8 memory cells. In conclusion, the inherent competition between two simultaneous immune responses results in significant alterations in T cell immunity and subsequent disease outcome upon reexposure. IMPORTANCE: Combination vaccines and simultaneous administration of vaccines are necessary to accommodate required immunizations and maintain vaccination rates. Antibody responses generally correlate with protection and vaccine efficacy. However, live attenuated vaccines also induce strong CD8 T cell responses, and the impact of these cells on subsequent immunity, whether beneficial or detrimental, has seldom been studied, in part due to the lack of known T cell epitopes to vaccine viruses. We questioned if the inherent increased competition and stochasticity between two immune responses during a simultaneous coinfection would significantly alter CD8 T cell memory in a mouse model where CD8 T cell epitopes are clearly defined. We show that some of the coinfected mice have sufficiently altered memory T cell responses that they have decreased protection and enhanced immunopathology when reexposed to one of the two viruses. These data suggest that a better understanding of human T cell responses to vaccines is needed to optimize immunization strategies

Clonal exhaustion as a mechanism to protect against severe immunopathology and death from an overwhelming CD8 T cell response

The balance between protective immunity and immunopathology often determines the fate of the virus-infected host. How rapidly virus is cleared is a function of initial viral load, viral replication rate, and efficiency of the immune response. Here, we demonstrate, with three different inocula of lymphocytic choriomeningitis virus (LCMV), how the race between virus replication and T cell responses can result in different disease outcomes. A low dose of LCMV generated efficient CD8 T effector cells, which cleared the virus with minimal lung and liver pathology. A high dose of LCMV resulted in clonal exhaustion of T cell responses, viral persistence, and little immunopathology. An intermediate dose only partially exhausted the T cell responses and resulted in significant mortality, and the surviving mice developed viral persistence and massive immunopathology, including necrosis of the lungs and liver. This suggests that for non-cytopathic viruses like LCMV, hepatitis C virus, and hepatitis B virus, clonal exhaustion may be a protective mechanism preventing severe immunopathology and death

mRNA-mediated glycoengineering ameliorates deficient homing of human stem cell-derived hematopoietic progenitors

Generation of functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) has been a long-sought-after goal for use in hematopoietic cell production, disease modeling, and eventually transplantation medicine. Homing of HSPCs from bloodstream to bone marrow (BM) is an important aspect of HSPC biology that has remained unaddressed in efforts to derive functional HSPCs from human PSCs. We have therefore examined the BM homing properties of human induced pluripotent stem cell-derived HSPCs (hiPS-HSPCs). We found that they express molecular effectors of BM extravasation, such as the chemokine receptor CXCR4 and the integrin dimer VLA-4, but lack expression of E-selectin ligands that program HSPC trafficking to BM. To overcome this deficiency, we expressed human fucosyltransferase 6 using modified mRNA. Expression of fucosyltransferase 6 resulted in marked increases in levels of cell surface E-selectin ligands. The glycoengineered cells exhibited enhanced tethering and rolling interactions on E-selectin-bearing endothelium under flow conditions in vitro as well as increased BM trafficking and extravasation when transplanted into mice. However, glycoengineered hiPS-HSPCs did not engraft long-term, indicating that additional functional deficiencies exist in these cells. Our results suggest that strategies toward increasing E-selectin ligand expression could be applicable as part of a multifaceted approach to optimize the production of HSPCs from human PSCs

Vaccine breakthrough hypoxemic COVID-19 pneumonia in patients with auto-Abs neutralizing type I IFNs

Life-threatening `breakthrough' cases of critical COVID-19 are attributed to poor or waning antibody response to the SARS- CoV-2 vaccine in individuals already at risk. Pre-existing autoantibodies (auto-Abs) neutralizing type I IFNs underlie at least 15% of critical COVID-19 pneumonia cases in unvaccinated individuals; however, their contribution to hypoxemic breakthrough cases in vaccinated people remains unknown. Here, we studied a cohort of 48 individuals ( age 20-86 years) who received 2 doses of an mRNA vaccine and developed a breakthrough infection with hypoxemic COVID-19 pneumonia 2 weeks to 4 months later. Antibody levels to the vaccine, neutralization of the virus, and auto- Abs to type I IFNs were measured in the plasma. Forty-two individuals had no known deficiency of B cell immunity and a normal antibody response to the vaccine. Among them, ten (24%) had auto-Abs neutralizing type I IFNs (aged 43-86 years). Eight of these ten patients had auto-Abs neutralizing both IFN-a2 and IFN-., while two neutralized IFN-omega only. No patient neutralized IFN-ss. Seven neutralized 10 ng/mL of type I IFNs, and three 100 pg/mL only. Seven patients neutralized SARS-CoV-2 D614G and the Delta variant (B.1.617.2) efficiently, while one patient neutralized Delta slightly less efficiently. Two of the three patients neutralizing only 100 pg/mL of type I IFNs neutralized both D61G and Delta less efficiently. Despite two mRNA vaccine inoculations and the presence of circulating antibodies capable of neutralizing SARS-CoV-2, auto-Abs neutralizing type I IFNs may underlie a significant proportion of hypoxemic COVID-19 pneumonia cases, highlighting the importance of this particularly vulnerable population

Anti-IFN-gamma and peptide-tolerization therapies inhibit acute lung injury induced by cross-reactive influenza A-specific memory T cells

Viral infections have variable outcomes, with severe disease occurring in only few individuals. We hypothesized that this variable outcome could correlate with the nature of responses made to previous microbes. To test this, mice were infected initially with influenza A virus (IAV) and in memory phase challenged with lymphocytic choriomeningitis virus (LCMV), which we show in this study to have relatively minor cross-reactivity with IAV. The outcome in genetically identical mice varied from mild pneumonitis to severe acute lung injury with extensive pneumonia and bronchiolization, similar to that observed in patients who died of the 1918 H1N1 pandemic. Lesion expression did not correlate with virus titers. Instead, disease severity directly correlated with and was predicted by the frequency of IAV-PB1703- and IAV-PA224-specific responses, which cross-reacted with LCMV-GP34 and LCMV-GP276, respectively. Eradication or functional ablation of these pathogenic memory T cell populations, using mutant-viral strains, peptide-based tolerization strategies, or short-term anti-IFN-gamma treatment, inhibited severe lesions such as bronchiolization from occurring. Heterologous immunity can shape outcome of infections and likely individual responses to vaccination, and can be manipulated to treat or prevent severe pathology

Vaccination and heterologous immunity: educating the immune system

This review discusses three inter-related topics: (1) the immaturity of the neonatal and infant immune response; (2) heterologous immunity, where prior infection history with unrelated pathogens alters disease outcome resulting in either enhanced protective immunity or increased immunopathology to new infections, and (3) epidemiological human vaccine studies that demonstrate vaccines can have beneficial or detrimental effects on subsequent unrelated infections. The results from the epidemiological and heterologous immunity studies suggest that the immune system has tremendous plasticity and that each new infection or vaccine that an individual is exposed to during a lifetime will potentially alter the dynamics of their immune system. It also suggests that each new infection or vaccine that an infant receives is not only perturbing the immune system but is educating the immune system and laying down the foundation for all subsequent responses. This leads to the question, is there an optimum way to educate the immune system? Should this be taken into consideration in our vaccination protocols

Humanized Mouse Models of Clinical Disease.

Immunodeficient mice engrafted with functional human cells and tissues, that is, humanized mice, have become increasingly important as small, preclinical animal models for the study of human diseases. Since the description of immunodeficient mice bearing mutations in the IL2 receptor common gamma chain (IL2rg(null)) in the early 2000s, investigators have been able to engraft murine recipients with human hematopoietic stem cells that develop into functional human immune systems. These mice can also be engrafted with human tissues such as islets, liver, skin, and most solid and hematologic cancers. Humanized mice are permitting significant progress in studies of human infectious disease, cancer, regenerative medicine, graft-versus-host disease, allergies, and immunity. Ultimately, use of humanized mice may lead to the implementation of truly personalized medicine in the clinic. This review discusses recent progress in the development and use of humanized mice and highlights their utility for the study of human diseases. Annu Rev Pathol 2017; 12:187-215