Browsing by Author "Permar, Sallie R"

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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.

A better understanding of 1) maternal HIV Envelope (Env)-specific IgG responses that are partially protective against vertical HIV transmission, and 2) factors that mediate the transplacental transfer of maternal protective IgG is needed to improve infant health in early life, in which maternal passively-acquired IgG mediates protection against neonatal infections. To understand maternal factors and IgG characteristics that mediate transplacental IgG transfer, we examined transplacental transfer efficiency determinants of maternal HIV and standard vaccine-antigen-specific IgG in a population of HIV-infected women, which have disrupted transplacental IgG transfer. Our findings suggest that maternal health factors and maternal IgG characteristics, such as binding to placentally expressed Fc receptors, IgG subclass frequency, and Fc region glycan profiles all mediate transplacental IgG transfer efficiency. We also identified maternal linear variable loop 3 (V3)-specific IgG binding and neutralizing responses targeting the C terminal region as partially protective against vertical transmission of HIV. These novel findings provide a roadmap of maternal factors and IgG characteristics as targets that can be harnessed to improve the transplacental IgG transfer of routinely-administered maternal vaccines and benchmarks for assessing maternal HIV vaccines that target the V3 loop.

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.

Human cytomegalovirus (HCMV) is a ubiquitous, host-restricted β-herpesvirus that has co-evolved with humans over millennia. In healthy individuals, HCMV infection is usually mild or potentially beneficial, as seropositivity can boost host heterologous immunity. Conversely, infection in immunocompromised individuals or in utero can cause severe disease. Congenital HCMV infection (cCMV), acquired via transplacental transmission from mother to fetus, is the most common vertically transmitted infection worldwide and is associated with stillbirth, neurodevelopmental impairment, hearing loss, and childhood leukemia. There are no vaccines or therapeutics to prevent cCMV and our knowledge of the impacts of infection on neonatal immunity remain limited. In my dissertation work, we aimed to discover protective immune responses against cCMV infection and to define how cCMV modulates neonatal immunity. To investigate these aims, we established a case-control cohort of mother-infant dyads with and without cCMV infection using the Carolinas Cord Blood Bank. We leveraged paired maternal and cord blood samples to define 1) how HCMV modifies placental IgG transfer, 2) protective antibody responses against cCMV transmission, and 3) the impact of cCMV on neonatal cellular immunity. First, we found that placental IgG transfer efficiency was reduced in cCMV infection because HCMV transmitting mothers had high rates of hypergammaglobulinemia, leading to saturation of the neonatal Fc receptor (FcRn) and decreased IgG transfer ratios. However, neonates with and without cCMV infection had similar levels of antigen-specific IgG, suggesting equivalent protection conferred by FcRn-mediated IgG transfer. When comparing HCMV transmitting to HCMV seropositive non-transmitting dyads, we observed that placental transfer of high avidity HCMV-specific IgG was reduced in cCMV infection but that cCMV-infected infants had high levels of neutralizing, HCMV-specific IgG. Next, we found that antibody-dependent cellular phagocytosis (ADCP), a Fc-mediated antibody effector function, was associated with protection against cCMV transmission. We determined that HCMV-specific IgG activation of FcγRI and FcγRIIA, which mediate ADCP and other innate immune cell effector functions by binding to the Fc region of IgG, was enhanced in mothers who did not transmit HCMV in utero. Finally, we compared cord blood immunophenotypes in infants with and without cCMV infection. We found that activated CD8+ and CD4+ T cells were increased and that regulatory T cells were reduced in cCMV infection. Moreover, we discovered that a unique population of “NKT-like” CD8+ T cells expressing NK cell markers such as FcγRIIIA/CD16 and NKG2A/C was significantly expanded in cord blood from cCMV-infected neonates. Cumulatively, our data highlights the important role of Fc-mediated immunity in cCMV infection and reveals that HCMV is a powerful immunomodulator in utero, which can guide future vaccination strategies against HCMV and other infectious diseases.

Zika virus (ZIKV) re-emerged in the 2015-2016 epidemic in the Americas, when it was recognized that this mosquito transmitted virus can also be transmitted congenitally. One in 10 infants born to ZIKV-infected pregnancies presented with congenital defects including microcephaly, brain and ocular damage, neurodevelopmental delays, and mobility defects. In the past outbreak 11,000 children in Brazil were born with microcephaly and even more have lifelong disability. To prevent congenital transmission, a vaccine that is effective in pregnancy is urgently needed. Developing such an intervention requires an understanding of the targeted immune responses that mediates protection in pregnancy. Therefore, we investigated maternal B cell and antibody immunity to ZIKV.

First, we characterized ZIKV immunity in mothers with natural ZIKV infection and found that all mounted high ZIKV neutralizing antibody titers within 10 days of symptoms and maintained this throughout gestation. We then evaluated transplacental transfer of IgG and found that maternal ZIKV infection did not impair transfer of vaccine-elicited and flavivirus-neutralizing IgG, indicating that maternal immunization is a viable option to transfer immune protection to the newborn. However, a potential risk of transferred IgG is antibody-dependent enhancement of a heterologous flavivirus in early life. Consequently, we next tested the role of vertically transferred cross-reactive IgG in infant cord blood and found that transferred IgG may mediate in vitro flavivirus enhancement in absence of high ZIKV-neutralizing titers.

Next, we evaluated the role of IgM antibodies in the maternal immune response to ZIKV, as IgM do not cross the placenta during gestation and were found for an unusually long time in many cohorts. We found that plasma IgM contributes to early ZIKV neutralization across several ZIKV-infected pregnant women. We then isolated a potently ZIKV-neutralizing IgM monoclonal antibody, DH1017.IgM, which demonstrates 39-fold increased neutralization activity than a recombinant IgG with the same antigen binding sites, suggesting that the multivalency of the IgM may have a role in function. Potency of DH1017.IgM increased in a dose-dependent manner with complement. Structural studies of the epitope revealed a novel E dimer epitope on Domain II. This epitope can be bound by DH1017.Fab at multiple angles and is computationally predicted to have the capacity to be bound by all five IgM monomers simultaneously, defining a novel route of ZIKV neutralization. Importantly, the DH017.IgM protects mice from a lethal challenge of ZIKV and reduced in vitro enhancement otherwise observed with DH1017.IgG, suggesting that DH1017.IgM may be a suitable candidate for prophylactic intervention in pregnancy.

Altogether, we have identified key aspects of maternal immunity that will inform the development of ZIKV vaccines. First, we show that pregnant women can mount a robust B cell response against ZIKV, that is durable throughout pregnancy, and that protective levels of vaccine-elicited IgG can be transferred transplacentally to infants, suggesting that maternal immunization is a viable strategy to prevent infections in pregnancy and protect the newborn. However, with the transfer of flavivirus cross-reactive IgG, there is a need to monitor infants for enhanced flavivirus disease upon maternal immunization. Subsequently, we found that IgM antibodies in plasma contribute to ZIKV neutralization and identified a novel route for virus neutralization by DH1017.IgM that can be leveraged in immunogen design. Thus, vaccine design should assess if inclusion of multimeric immunogens that mimic the conformational surface of the virion supports development of IgM-mediated immunity.