Browsing by Subject "Cytomegalovirus"

Human cytomegalovirus (HCMV) is the most common cause of congenital infection worldwide, impacting 1 in 150 live-born infants. Children afflicted by congenital HCMV frequently suffer from lifelong, debilitating neurologic sequelae including microcephaly, sensorineural hearing loss, and cognitive impairment. Natural maternal immunity to HCMV reduces the frequency of congenital infection, but does not prevent the disease altogether. Thus, a vaccine to reduce the incidence and severity of infant infection is a public health priority. Employing a nonhuman primate model of congenital CMV transmission as well as clinical samples from a partially-efficacious HCMV vaccine trial, we sought to examine both the attributes of anti-HCMV immune responses that provide protective immunity as well as the impact of vaccine-elicited immunity on the in vivo HCMV viral population.

First, we used a nonhuman primate model of congenital CMV infection to investigate the ability of preexisting antibodies to protect against placental CMV transmission in the setting of primary maternal infection and subsequent viremia, which is required for placental virus exposure. Pregnant, CD4+ T cell-depleted, rhesus CMV (RhCMV)-seronegative rhesus monkeys were treated with either standardly-produced hyperimmune globulin (HIG) from RhCMV-seropositive macaques or dose-optimized, potently RhCMV-neutralizing HIG prior to intravenous challenge with an RhCMV swarm. HIG passive infusion provided complete protection against fetal loss in both groups. The dose-optimized, RhCMV-neutralizing HIG additionally inhibited placental transmission of RhCMV and reduced viral replication and diversity. Our findings suggest that the presence of durable and potently-neutralizing antibodies at the time of primary infection can prevent transmission of systemically-replicating maternal RhCMV to the developing fetus.

Next, we assessed the properties of antibody responses elicited by glycoprotein B (gB) + MF59 adjuvant subunit vaccination – the most successful HCMV vaccine tested clinicaly to-date, which demonstrated approximately 50% efficacy in preventing HCMV acquisition in multiple phase 2 trials. Plasma from 33 gB/MF59 vaccinees at peak immunogenicity was tested for gB epitope specificity as well as neutralizing and non-neutralizing anti-HCMV effector functions, and compared to an HCMV-seropositive cohort. gB/MF59 vaccination elicited IgG responses with gB-binding magnitude and avidity comparable to natural infection. Additionally, IgG subclass distribution was similar with predominant IgG1 and IgG3 responses induced by gB vaccination and HCMV infection. However, vaccine-elicited antibodies exhibited limited neutralization of the autologous virus, negligible neutralization of multiple heterologous strains, and limited binding responses against gB structural motifs targeted by neutralizing antibodies including AD-1, AD-2, and Domain I. Interestingly, vaccinees had high-magnitude IgG responses against AD-3 linear epitopes, demonstrating immunodominance against this non-neutralizing, cytosolic region. Finally, vaccine-elicited IgG robustly bound trimeric, membrane-associated gB on the surface of transfected or HCMV-infected cells and mediated virion phagocytosis, raising the possibility that non-neutralizing antibody effector functions contributed to the partial protection against HCMV acquisition observed in gB/MF59 vaccinees.

Lastly, we evaluated the impact of gB/MF59-elicited immune responses on the population of viruses acquired by trial participants. In this analysis, we employed quantitative PCR as well as two distinct next-generation sequencing strategies (short amplicon and whole gene) to interrogate genetic differences between the HCMV populations infecting gB/MF59 vaccinees and placebo recipients. For the majority of subject-specific viral populations analyzed, we identified 1 or 2 dominant viral variants, as well as a large number of minor variants present at very low frequency. This finding suggests that the intrahost viral population constitutes a heterogeneous swarm of genetically-distinct virus quasi-species. Additionally, we identified several distinctions between the viral populations of acutely-infected vaccinees and placebo recipients. First, there was reduced magnitude viral shedding in the saliva of gB vaccinees compared to placebo. Furthermore, we noted evidence of genetic compartmentalization at the gB locus in 3 of 4 vaccinees, though only in 1 of 7 placebo recipients. Finally, we observed an enrichment of gB1 genotype HCMV variants among placebo recipients compared to vaccinees, and hypothesize that the gB1 genotype vaccine immunogen might have elicited genotype-specific protection that accounts for the efficacy observed in clinical trial.

Thus, we have made several observations that will inform rational design of the next generation of HCMV vaccines. First, our data suggests that preexisting antibodies can protect against congenital CMV transmission in a rhesus monkey model, and thus that antibodies could be a primary target of vaccines to eliminate congenital HCMV infection. Secondly, our analysis of antibody responses elicited by gB/MF59 vaccination indicates that non-neutralizing antibody functions contributed to the observed 50% vaccine protection and therefore should be a consideration in future vaccine design. Finally, our examination of viral populations in gB/MF59 vaccinees indicates that gB-elicited antibodies had a measurable impact on viral intrahost population dynamics and that gB immunogen strain-specific responses may have defined vaccine protection, suggesting that immunogen strain breadth may be an important factor to consider for future vaccine design.

Human CMV (HCMV), a ubiquitous β-herpesvirus, remains the most common congenital infection and infectious complication in immunocompromised patients. Despite the severe clinical impacts, there is no current approved HCMV vaccine or immunotherapy. Two major hurdles have been identified for the HCMV vaccine development. First, the HCMV genome is highly diverse and variable, i.e., a HCMV vaccine design that only includes immunogens from a single strain might not provide effective protection against other HCMV strains. Second, a large gap remains to understand what virologic determinants are essential for congenital CMV transmission.

HCMV glycoprotein B (gB) and pentameric glycoprotein complex (PC) are currently the most promising vaccine targets. gB is essential for viral entry into all host cells and was shown to elicit both neutralizing and non-nAb responses. The most successful HCMV vaccine to-date, a gB subunit vaccine adjuvanted with MF59, achieved 50% efficacy against primary HCMV infection. The gB/MF59 vaccinees were less frequently infected with HCMV gB genotype strains most similar to the vaccine strain than strains encoding genetically distinct gB genotypes, suggests that the strain-specific immunity might account for the limited efficacy. Applying the lipid nanoparticles-encapsulated nucleoside-modified mRNA (mRNA-LNP) vaccine platform, we hypothesized that vaccination with multiple HCMV gB genotypes could increase the breadth of gB-specific humoral and cellular responses, leading to broader protection.

To test our hypothesis, we intradermally immunized female rabbits with three doses of the monovalent or multivalent gB mRNA-LNP vaccines and measured the vaccine-elicited humoral and cellular responses. Compared to the monovalent vaccine, the multivalent vaccines did not demonstrate a higher magnitude or breadth of gB-specific IgG binding or functional antibody responses against multiple gB genotype. In addition, the multivalent vaccines did not elicit a stronger T cell response against variable regions among gB genotypes. Our data suggests that inclusion of multivalent gB antigens is not an effective strategy to increase the breadth of anti-HCMV gB antibody and T cell responses.

PC, composed of the subunits gH/gL/UL128/UL130/UL131A, has been demonstrated to be essential for CMV entry into non-fibroblast cells in vitro. The PC was also identified as the major target of neutralizing antibodies. As HCMV infection of most cell types found near the maternal-fetal interface depends on the PC-mediated entry in vitro, it is conceivable that this complex could be required for cross-placental CMV transmission in vivo. These findings link the PC to broad cell tropism and virus dissemination in vivo, denoting all subunits as potential targets for intervention strategies and vaccine development.

To determine the importance of the PC for congenital transmission in a translational non-human primate (NHP) model, we engineered a rhesus CMV (RhCMV) mutant lacking the homologues of UL128 and UL130 which demonstrated diminished infection of epithelial cell in vitro. Nevertheless, intravenous inoculation of immunocompetent and CD4+ T cell-depleted, RhCMV-seronegative, pregnant rhesus macaques with the PC-deficient mutant resulted in similar maternal RhCMV peak plasma viremia levels to that of PC-intact RhCMV, while virus shedding in saliva and urine was limited. Infections with the PC-intact virus induced strong IgG responses that were able to neutralize RhCMV entry into epithelial cells. These responses were reduced, but not absent, from animals infected with the PC-deficient mutant, which also induced IgG responses against gH. Most importantly, congenital infection rates determined by the viral DNA detection in amniotic fluid was identical between the PC-deficient and PC-intact RhCMV. Our data indicates that the PC is dispensable for transplacental transmission in non-human primates.

In the past 30 years, the global prevalence of chronic respiratory diseases has increased by 40%. Importantly, lung transplantation remains the only definitive treatment for patients with advanced lung disease. Despite the rapidly growing need for lung transplantation, lung transplant recipients have by far the worst survival rates among all solid organ transplant types, with a current median survival of only 6 years post transplantation. Chronic lung allograft dysfunction (CLAD) is the leading cause of limited long-term survival of lung transplant recipients, affecting approximately 50% of patients within 5 years post-transplantation. Previous studies have identified humoral alloimmune responses against mismatched donor HLA and cytomegalovirus (CMV) infection as primary independent risk factors for chronic deterioration of the transplanted allograft and the fatal diagnosis of CLAD. This dissertation is comprised of work that aims to address critical gaps in knowledge in the fields of transplant immunology and infectious disease. The first component focuses on CMV-specific humoral immunity in naturally infected healthy blood donors (Chapter 2) and in CMV seropositive lung transplant recipients (Chapter 3). The second area of study focuses on humoral alloimmune responses and current methods used to evaluate and characterize donor HLA-specific alloantibodies (Chapter 4), as well as factors contributing to HLA-DQ immunogenicity (Chapter 5). These projects utilized clinical data and longitudinally collected human plasma samples from the completed multicenter Clinical Trials in Organ Transplantation (CTOT)-20 and CTOT-22 consortium studies. The findings from Chapter 2 contribute to our knowledge of naturally acquired immunity against CMV, which informs strategies for antibody-based therapeutics to help treat CMV infection as well as CMV vaccine development. The results from Chapter 3 indicate that CMV-specific antibodies have the potential to be used as novel biomarkers for viremia risk, possibly in combination with other known risk factors for viremia including CMV-specific T cell responses. These tools may help inform antiviral treatment duration in clinical practice. Chapter 4 of this dissertation represents the first in-depth technical comparison across traditional and modified single antigen bead Luminex assays using IgG enriched sera for donor-HLA specific post-transplant monitoring. These findings may be useful for clinical workflows to help improve efficiency and cost by stratifying patient samples that may benefit from additional testing in modified assays. Finally, Chapter 5 of this dissertation examines the specificity and timing of donor HLA-specific antibodies (DSA), as well as other risk factors for DSA development. In this project, we detail novel strategies for calculating HLA-DQ mismatch by accounting for trans-encoded DQ heterodimer formation. The results from this study also reveal elevated risk for developing DSA among patients with high-risk epitope mismatches and suggest that different organs may require different HLA mismatch calculation strategies. Overall, the results from these projects advance our understanding of the human immune response to alloantigens and cytomegalovirus in lung transplantation and push the field forward to help improve patient outcomes.

Background

Methods

Results

Conclusions

Glioblastoma (GBM) is a grade IV astrocytoma in which the median overall survival is approximately 15 months at time of diagnosis. Even with the current multi- modal therapeutic approach of surgery, chemotherapy with the DNA alkylating agent temozolomide, and radiation therapy, GBM remains uniformly lethal. Immunotherapeutic interventions are a burgeoning field in many different cancer treatments. They offer the exquisite specificity endowed by the immune system with minimal toxicities and new methods are being developed to enhance the endogenous immune responses.

With the recent identification of human cytomegalovirus (CMV) present within glioblastoma tissue but void in the surrounding normal healthy parenchyma there have been significant efforts aimed at understanding the biologic implications of the presence of the virus within GBM tissues with preliminary work demonstrating several capabilities of the virus to enhance the oncogenic process.

Likewise, a key area of importance in the development and design of effective immunotherapeutic platforms is the identification and targeting of tumor-specific antigens. The success of any immunotherapy platform relies heavily on the ability to selectively target antigens present within tumors but absent on healthy tissue, regardless of its role in tumorigenesis, as well as having robust immunogenic properties.

CMV offers a plethora of possible targets, as it is the largest known DNA virus that infects humans, yet very little is known about its biological significance in glioblastoma pathogenesis as well as the most efficacious and immunogenic targets for immunotherapeutic development.

We have been able to elucidate more thoroughly the feasibility and potency of an immunologic platform targeting CMV within glioblastoma utilizing a multi-antigen multi-component peptide based strategy that demonstrated significant immunogenicity and anti-tumor activity in pre-clinical models utilizing various assays. We have also developed several sensitive and specific detection methodologies including: 1) custom gene expression microarrays, 2) multiplex real time quantitative polymerase chain reaction (RT-qPCR) assays, 3) a massively parallel RNA deep sequencing platform, and 4) immunological assays. We have also successfully determined the capacity for endogenous CMV gene expression to be maintained in primary glioblastoma cell lines as well as examining the preponderance of CMV gene expression in a subpopulation of glioma stem cell-like cells, the slow cycling GBM cells established from primary tumor tissues, in an attempt to illuminate some of the biologic underpinnings of CMV with respect to GBM pathogenesis.

Taken together, these data lay the groundwork for the development of a more efficacious vaccination strategy targeting CMV in GBM. The screening strategies employed throughout this work will allow for an accurate antigenic profile of CMV in GBM which will subsequently permit the design of a more robust peptide vaccine for the next generation of cancer vaccine. We have also begun to describe some of the interesting biologic phenomena associated with CMV in GBM, as our results demonstrate continued viral gene expression in glioma stem cell-like cell populations indicating viral tropism for certain cell types.

Background

Methods

Results

Conclusions

Human cytomegalovirus (CMV) is the most common cause of congenital infection worldwide, affecting approximately 1 in 150 infants, and is a leading cause of morbidity and mortality among transplant recipients. Congenital CMV (cCMV) infection can lead to permanent hearing loss, brain damage, and neurodevelopmental delay, and cCMV alone is responsible for nearly 25% of all infant hearing loss. Chronic CMV infection has also been associated with a heightened inflammatory state and increased risk of aging-related diseases, including as cardiovascular disease and type 2 diabetes. Over the last fifty years, there have been many efforts to develop vaccines that can prevent CMV disease. However, vaccine development for CMV faces many challenges, including a limited understanding of the immune responses protective against infection.

Like other beta herpesviruses, CMV can establish a lifelong, persistent infection in hosts, marked by periods of latency and reactivation, and preexisting immunity does not protect against reinfection. Moreover, CMV is known to predominantly spread by direct cell-to-cell transmission, complicating efforts to design vaccines that not only prevent viral entry via antibody neutralization but also inhibit the spread of cell-associated virus. To identify targets for vaccine development, we investigated the antibody immune responses associated with protection from CMV in historical vaccine trials and the lineage maturation of neutralizing antibodies elicited in natural infection.

The most efficacious CMV vaccine to-date is the glycoprotein B (gB) subunit vaccine combined with the MF59 adjuvant (gB/MF59), which achieved 50% protection against primary CMV acquisition in multiple Phase 2 clinical trials. CMV gB is a viral envelope protein that mediates fusion with host cell membranes and is required for viral entry into all known cell types and for cell-cell spread. Previous studies had found that in cohorts of CMV-seronegative postpartum women and renal transplant recipients, gB/MF59 vaccination did not elicit broadly neutralizing antibodies but instead generated robust nonneutralizing antibody responses, namely antibody-dependent cellular phagocytosis (ADCP). These studies lacked the statistical power to determine whether ADCP responses contributed to the partial efficacy of the gB/MF59 vaccine. We aimed to define the immune responses correlated with protection from primary CMV acquisition in Phase 2 gB/MF59 clinical trials in cohorts of adolescent girls and postpartum women. We first evaluated the vaccine-elicited sera IgG binding, neutralizing, and nonneutralizing responses against CMV, and we observed distinct immunogenicity profiles in the adolescent and postpartum cohorts, wherein adolescent vaccinees but not postpartum vaccinees developed broadly neutralizing CMV antibodies. We then compared the sera antibody responses between vaccinees who acquired infection and those who remained uninfected during the course of the trial. By multiple logistic regression analysis, we found that protection against primary CMV acquisition was associated with the presence of sera IgG binding to cell-associated gB, but not IgG binding to soluble gB as used in the gB/MF59 vaccine. These results suggested that there may be conformational differences between cell-associated and soluble gB. Supporting this, we identified gB-specific monoclonal antibodies (mAbs) that differentially recognized these gB structures. Our findings indicated the importance of the native, cell-associated gB conformation in future CMV vaccine design.

In our immunogenicity studies of gB/MF59, we observed that the vaccine failed to elicit antibodies against the gB antigenic domain 2 site 1 region (AD-2S1), which is a highly conserved, linear epitope at the far N terminus of gB that is known to be a target for potently neutralizing antibodies in natural infection. The presence of sera antibodies against gB AD-2S1 in naturally infected individuals have been associated with decreased risk for cCMV transmission and less severe CMV disease in transplant recipients. Yet, only about half of naturally infected individuals develop anti-gB AD-2S1 antibodies, and gB-based vaccines to-date have failed to elicit these responses. Thus, it remained unclear how to generate neutralizing antibodies against this poorly immunogenic epitope by vaccination. With the goal to identify a gB AD-2S1 structure that could elicit these potently neutralizing antibodies from the germline, we employed a B cell lineage-targeted vaccine strategy. We mapped the phylogeny of a well-characterized, potently neutralizing anti-gB AD-2S1 mAb from its germline precursor, then by empiric testing, we identified the antibody mutations that conferred neutralizing function. We found that a single heavy chain mutation in the CDR1 region was both necessary and sufficient to confer neutralizing function to the otherwise nonneutralizing IGHV3-30/IGKV3-11 germline ancestor mAb. This study identified a critical, early B cell receptor mutation that can serve a target for mutation-guided CMV vaccine design.

Only a limited number of gB AD-2S1 mAb sequences have been published to-date, with few clonally related members. To identify additional antibodies for lineage analysis and for evaluation as potential passive therapeutics, we developed a pipeline to sequence anti-CMV gB AD-2S1 mAbs and estimate their antigen binding in high-throughput. This study leveraged modern advances in B cell sequencing technologies to expedite the discovery of potently neutralizing CMV antibodies.

In sum, this work contributes to our understanding of protective immunity to CMV. We found that the immune correlate of protection for the gB/MF59 vaccine was sera IgG binding to cell-associated gB, suggesting that the next generation of gB-based vaccines should be designed to adopt a cell-associated or native conformation. Then, we investigated the lineage maturation of potently neutralizing antibodies against CMV gB AD-2S1 and identified a critical mAb mutation from the germline associated with the development of neutralization function, which will may serve as target for immunogen design. Additionally, we developed a pipeline for the high-throughput isolation of anti-gB AD-2S1 mAbs. These findings provide insight into antibody-mediated protection from CMV and illuminate paths forward for rational vaccine design.

Cytomegalovirus (CMV) is the most common congenital infection and a problematic opportunistic pathogen for immunocompromised patient populations. Despite the immense global burden of CMV and many years of research, the licensed interventions for prevention of CMV disease, and congenital CMV in particular, are very limited. The goal of the work presented in this dissertation is to inform vaccine design for the prevention of congenital CMV by enhancing maternal humoral immunity. We initially investigated the humoral immune responses that associate with protection from vertical CMV transmission in a rhesus macaque model of congenital CMV, evaluating Fc mediated effector responses, which had not previously been measured in the context of rhesus CMV (RhCMV) infection. This study suggested that the humoral response develops too late following primary infection to play a significant role in prevention of vertical transmission but demonstrated a role for pre-existing, potently neutralizing antibodies in prevention of vertical CMV transmission. While this study did not find an association between Fc mediated antibody effector responses, clinical observational studies have implicated these antibody functions in protection from congenital CMV. Interestingly, human CMV (HCMV) is known to encode multiple proteins capable of binding to immunoglobulin G (IgG) antibodies, which have demonstrated the ability to interfere with host Fcγ receptor (FcγR) activation and effector function. We have identified homolog viral FcγRs (vFcγRs) in RhCMV, which allows us a unique opportunity to study these proteins in vivo for the first time by infecting RhCMV-seronegative rhesus macaques with RhCMV lacking all three identified vFcγRs and validating the role vFcγRs have demonstrated in vitro in immune evasion. Lastly, we evaluated a novel HCMV vaccine strategy in which we targeted this immune evasion mechanism through active vaccination against glycoprotein B (gB) alone or in combination with one of the vFcγRs. This proof-of-concept study demonstrated that immune responses against the vFcγRs, gp34 in particular, can improve host FcγR activation and effector function, with initial focus on FcRI (CD64). These results suggest that a simple addition of one or more vFcγRs to vaccines already in development may have a significant impact on the effectiveness of Fc mediated effector responses, which could in turn reduce the risk of vertical CMV transmission.

This dissertation focuses on applications of partial differential equations with an emphasis on chemotaxis. Chemotaxis is a response of motile cells or organisms in which the direction of movement is affected by the gradient of a diffusible substance. In multicellular organisms, chemotaxis is critical to development as well as normal function. We also include a specific application that utilizes differential equations and stochastic simulation to model placental transmission of Cytomegalovirus (CMV) with experimental data.Our methods include regularity estimates, maximal and comparison principles, probabilistic techniques, and stochastic processes. This work resulted in several conclusions: We prove quantitatively chemotaxis enhances reaction in 1D setting: the upper bound for reaction time with chemotaxis is smaller than the lower bound for reaction time without chemotaxis.

We have also analyzed a random searcher in shear flow and chemotaxis; on the rigorous level, we are able to establish that the very large shear rates are a dimension reduction mechanism: the expected search time converges to the one of the corresponding one-dimensional problem. Numerically, we see fast decrease of the expected hitting time for shear and chemotactic coupling; we discover there is an optimal shear rate range where the searching time is minimal.

We also explore a model aiming to describe the origin of chemotactic ability of cells, in particular proving global regularity.

We present an end-to-end model of placental CMV transmission that allow us to study different type of infections, the timing of inoculation, the effect of immune suppression on the risk of placental transmission, and the effect of treatment.

The evolution of antigenically variable viruses cannot be understood without studying the interaction between viruses and the host immune system. Viral evolution is driven by their fast acquisition of genetic variation as well as by the strong selection imposed by the host immune response. Moreover, understanding viral evolution dynamics and its interplay on the host immune response can provide essential information for vaccine development. In this dissertation, I use an integrative approach to study various aspects of this interplay in two viral systems: influenza A (IAV) and cytomegalovirus (CMV), both ubiquitous in humans and significant public health threats.

Congenital cytomegalovirus infection is the leading infectious cause of congenital defects. As such, the study of viral dynamics is essential to develop better treatment and prevention procedures. In a monkey challenge study for congenital cytomegalovirus infection, I investigated viral transmission between maternal and fetal compartments. Using high-coverage sequencing data, I examined viral evolutionary dynamics in time and space. I found evidence of large transmission bottleneck sizes between maternal compartments and in congenital transmission. I also inquire about the role of preexistent CMV-specific antibodies in the virus population, finding no apparent effect in the viral genetic make-up but reduced viral load and also reduced congenital transmission.

One of the more promising vaccine formulation for CMV until now is the gB-MF59 vaccine, which is based on a soluble version of the immunodominant gB protein. To understand immune and viral factors contributing to vaccine efficacy for this formulation, I examined immunoglobulin G binding to a gB-specific peptide microarray from seropositive individuals and vaccinees prior to and after vaccination. The antibody profile observed from binding clustered by individual immune exposure history. While the antibody profile elicited by vaccination show high agreement with the one from seropositive individuals, I also identified regions in gB preferentially targeted in vaccinees. Moreover, I observe no difference between the antibody profiles of vaccinees with different clinical outcomes and instead found further evidence for reduced cross-immunity between divergent genotypes.

Original antigenic sin (OAS) refers to the tendency of the host immune system to focus on previously recognized viral epitopes during secondary challenges with related viral strains. This preference is sustained by antibody memory and can result in suboptimal immune protection. Mounting evidence highlights the importance of the initial viral strain encountered during childhood, which primes the antibody repertoire to contend against further infections. Here, the goal was to identify OAS-driven cohort effects in IAV driven by at least one antigenic mutation. Using sequence data and host's age information from individuals infected with H1N1 in the U.S. from 2009 to the present, we searched for potential signatures of birth year cohort changes driven by new variants with nonsynonymous mutations. We identified multiple variants with such properties and studied the age groups with differential abundance after such variant arose.