High-dimensional analysis of 16 SARS-CoV-2 vaccine combinations reveals lymphocyte signatures correlating with immunogenicity.

The range of vaccines developed against severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) provides a unique opportunity to study immunization across different platforms. In a single-center cohort, we analyzed the humoral and cellular immune compartments following five coronavirus disease 2019 (COVID-19) vaccines spanning three technologies (adenoviral, mRNA and inactivated virus) administered in 16 combinations. For adenoviral and inactivated-virus vaccines, heterologous combinations were generally more immunogenic compared to homologous regimens. The mRNA vaccine as the second dose resulted in the strongest antibody response and induced the highest frequency of spike-binding memory B cells irrespective of the priming vaccine. Priming with the inactivated-virus vaccine increased the SARS-CoV-2-specific T cell response, whereas boosting did not. Distinct immune signatures were elicited by the different vaccine combinations, demonstrating that the immune response is shaped by the type of vaccines applied and the order in which they are delivered. These data provide a framework for improving future vaccine strategies against pathogens and cancer.

HLA-DR15 Molecules Jointly Shape an Autoreactive T Cell Repertoire in Multiple Sclerosis.

The HLA-DR15 haplotype is the strongest genetic risk factor for multiple sclerosis (MS), but our understanding of how it contributes to MS is limited. Because autoreactive CD4+ T cells and B cells as antigen-presenting cells are involved in MS pathogenesis, we characterized the immunopeptidomes of the two HLA-DR15 allomorphs DR2a and DR2b of human primary B cells and monocytes, thymus, and MS brain tissue. Self-peptides from HLA-DR molecules, particularly from DR2a and DR2b themselves, are abundant on B cells and thymic antigen-presenting cells. Furthermore, we identified autoreactive CD4+ T cell clones that can cross-react with HLA-DR-derived self-peptides (HLA-DR-SPs), peptides from MS-associated foreign agents (Epstein-Barr virus and Akkermansia muciniphila), and autoantigens presented by DR2a and DR2b. Thus, both HLA-DR15 allomorphs jointly shape an autoreactive T cell repertoire by serving as antigen-presenting structures and epitope sources and by presenting the same foreign peptides and autoantigens to autoreactive CD4+ T cells in MS.

Autophagy and autophagy-related proteins in the immune system.

Autophagy is an intracellular bulk degradation system that is highly conserved in eukaryotes. The discovery of autophagy-related ('ATG') proteins in the 1990s greatly advanced the mechanistic understanding of autophagy and clarified the fact that autophagy serves important roles in various biological processes. In addition, studies have revealed other roles for the autophagic machinery beyond autophagy. In this Review, we introduce advances in the knowledge of the roles of autophagy and its components in immunity, including innate immunity, inflammatory responses and adaptive immunity.

Degrade to stay healthy-Proteolytic interplay during inflammation.

Proteasomes and autophagy constitute the 2 main proteolytic machineries for cytoplasmic content. A new study in PLOS Biology now demonstrates that autophagy stimulation alters proteasome composition, degrading hyperactive immunoproteasomes and thereby limiting inflammation.

Natural killer cell responses to human oncogenic γ-herpesvirus infections.

The two γ-herpesviruses Epstein Barr virus (EBV) and Kaposi sarcoma associated herpesvirus (KSHV) are each associated with more than 1% of all tumors in humans. While EBV establishes persistent infection in nearly all adult individuals, KSHV benefits from this widespread EBV prevalence for its own persistence. Interestingly, EBV infection expands early differentiated NKG2A+KIR- NK cells that protect against lytic EBV infection, while KSHV co-infection drives accumulation of poorly functional CD56-CD16+ NK cells. Thus persistent γ-herpesvirus infections are sculptors of human NK cell repertoires and the respectively stimulated NK cell subsets should be considered for immunotherapies of EBV and KSHV associated malignancies.

The autophagy machinery interacts with EBV capsids during viral envelope release.

Autophagy serves as a defense mechanism against intracellular pathogens, but several microorganisms exploit it for their own benefit. Accordingly, certain herpesviruses include autophagic membranes into their infectious virus particles. In this study, we analyzed the composition of purified virions of the Epstein-Barr virus (EBV), a common oncogenic γ-herpesvirus. In these, we found several components of the autophagy machinery, including membrane-associated LC3B-II, and numerous viral proteins, such as the capsid assembly proteins BVRF2 and BdRF1. Additionally, we showed that BVRF2 and BdRF1 interact with LC3B-II via their common protein domain. Using an EBV mutant, we identified BVRF2 as essential to assemble mature capsids and produce infectious EBV. However, BdRF1 was sufficient for the release of noninfectious viral envelopes as long as autophagy was not compromised. These data suggest that BVRF2 and BdRF1 are not only important for capsid assembly but together with the LC3B conjugation complex of ATG5-ATG12-ATG15L1 are also critical for EBV envelope release.

Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

MHC class II-deficient mice allow functional human CD4+ T-cell development.

Humanized mouse models have been developed to study cell-mediated immune responses to human pathogens in vivo. How immunocompetent human T cells are selected in a murine thymus in such humanized mice remains poorly explored. To gain insights into this mechanism, we investigated the differentiation of human immune compartments in mouse MHC class II-deficient immune-compromised mice (humanized Ab0 mice). We observed a strong reduction in human CD4+ T-cell development but despite this reduction Ab0 mice had no disadvantage during Epstein-Barr virus (EBV) infection. Viral loads were equally well controlled in humanized Ab0 mice compared to humanized NSG mice, and improved T-cell recognition of autologous EBV-transformed B cells was observed, especially with respect to cytotoxicity. MHC class II blocking experiments with CD4+ T cells from humanized Ab0 mice demonstrated MHC class II restriction of lymphoblastoid cell line recognition. These findings suggest that a small number of CD4+ T cells in humanized mice can be solely selected on human MHC class II molecules, presumably expressed by reconstituted human immune cells, leading to improved effector functions.

Live Long and Prosper for Antigen Cross-Presentation.

Even though proteolytic antigen fragments are displayed for cross-presentation, rapid proteolysis of endocytosed antigens inhibits this process. In this issue of Immunity, Alloatti et al. (2015) describe how maturing dendritic cells keep phagosomes and lysosomes apart to ensure extended antigen life that leads to prosperous cross-presentation.

Targeted delivery of a vaccine protein to Langerhans cells in the human skin via the C-type lectin receptor Langerin.

Human skin is a preferred vaccination site as it harbors multiple dendritic cell (DC) subsets, which display distinct C-type lectin receptors (CLR) that recognize pathogens. Antigens can be delivered to CLR by antibodies or ligands to boost antigen-specific immune responses. This concept has been established in mouse models but detailed insights into the functional consequences of antigen delivery to human skin DC in situ are sparse. In this study, we cloned and produced an anti-human Langerin antibody conjugated to the EBV nuclear antigen 1 (EBNA1). We confirmed specific binding of anti-Langerin-EBNA1 to Langerhans cells (LC). This novel LC-based vaccine was then compared to an existing anti-DEC-205-EBNA1 fusion protein by loading LC in epidermal cell suspensions before coculturing them with autologous T cells. After restimulation with EBNA1-peptides, we detected elevated levels of IFN-γ- and TNF-α-positive CD4+ T cells with both vaccines. When we injected the fusion proteins intradermally into human skin explants, emigrated skin DC targeted via DEC-205-induced cytokine production by T cells, whereas the Langerin-based vaccine failed to do so. In summary, we demonstrate that antibody-targeting approaches via the skin are promising vaccination strategies, however, further optimizations of vaccines are required to induce potent immune responses.

Immune checkpoints in T cells during oncogenic γ-herpesvirus infections.

Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) are two persistent oncogenic γ-herpesviruses with an exclusive tropism for humans. They cause cancers of lymphocyte, epithelial and endothelial cell origin, such as Burkitt's and Hodgkin's lymphoma, primary effusion lymphoma, nasopharyngeal carcinoma, and Kaposi sarcoma. Mutations in immune-related genes but also adverse events during immune checkpoint inhibition in cancer patients have revealed molecular requirements for immune control of EBV and KSHV. These include costimulatory and coinhibitory receptors on T cells that are currently explored or already therapeutically targeted in tumor patients. This review discusses these co-receptors and their influence on EBV- and KSHV-associated diseases. The respective studies reveal surprising specificities of some of these receptors for immunity to these tumor viruses, benefits of their blockade for some but not other virus-associated diseases, and that EBV- and KSHV-specific immune control should be monitored during immune checkpoint inhibition to prevent adverse events that might be associated with their reactivation during treatment.

CD27 is required for protective lytic EBV antigen-specific CD8+ T-cell expansion.

Primary immunodeficiencies in the costimulatory molecule CD27 and its ligand, CD70, predispose for pathologies of uncontrolled Epstein-Barr virus (EBV) infection in nearly all affected patients. We demonstrate that both depletion of CD27+ cells and antibody blocking of CD27 interaction with CD70 cause uncontrolled EBV infection in mice with reconstituted human immune system components. While overall CD8+ T-cell expansion and composition are unaltered after antibody blocking of CD27, only some EBV-specific CD8+ T-cell responses, exemplified by early lytic EBV antigen BMLF1-specific CD8+ T cells, are inhibited in their proliferation and killing of EBV-transformed B cells. This suggests that CD27 is not required for all CD8+ T-cell expansions and cytotoxicity but is required for a subset of CD8+ T-cell responses that protect us from EBV pathology.

PLK1-dependent phosphorylation restrains EBNA2 activity and lymphomagenesis in EBV-infected mice.

While Epstein-Barr virus (EBV) establishes a life-long latent infection in apparently healthy human immunocompetent hosts, immunodeficient individuals are at particular risk to develop lymphoproliferative B-cell malignancies caused by EBV. A key EBV protein is the transcription factor EBV nuclear antigen 2 (EBNA2), which initiates B-cell proliferation. Here, we combine biochemical, cellular, and in vivo experiments demonstrating that the mitotic polo-like kinase 1 (PLK1) binds to EBNA2, phosphorylates its transactivation domain, and thereby inhibits its biological activity. EBNA2 mutants that impair PLK1 binding or prevent EBNA2 phosphorylation are gain-of-function mutants. They exhibit enhanced transactivation capacities, accelerate the proliferation of infected B cells, and promote the development of monoclonal B-cell lymphomas in infected mice. Thus, PLK1 coordinates the activity of EBNA2 to attenuate the risk of tumor incidences in favor of the establishment of latency in the infected but healthy host.

Distinctive Kaposi Sarcoma-Associated Herpesvirus Serological Profile during Acute Plasmodium falciparum Malaria Episodes.

The seroprevalence of Kaposi sarcoma-associated herpesvirus (KSHV) and the incidence of endemic Kaposi sarcoma (KS) overlap with regions of malaria endemicity in sub-Saharan Africa. Multiple studies have shown an increased risk of KSHV seroconversion in children from high malaria compared to low malaria regions; however, the impact of acute episodes of Plasmodium falciparum (P. falciparum) malaria on KSHV's biphasic life cycle and lytic reactivation has not been determined. Here, we examined KSHV serological profiles and viral loads in 134 children with acute malaria and 221 healthy children from high malaria regions in Kisumu, as well as 77 healthy children from low malaria regions in Nandi. We assayed KSHV, Epstein-Barr virus (EBV), and P. falciparum malaria antibody responses in these three by multiplexed Luminex assay. We confirmed that KSHV seroprevalence was significantly associated with malaria endemicity (OR = 1.95, 1.18-3.24 95% CI, p = 0.01) with 71-77% seropositivity in high-malaria (Kisumu) compared to 28% in low-malaria (Nandi) regions. Furthermore, KSHV serological profiles during acute malaria episodes were distinct from age-matched non-malaria-infected children from the same region. Paired IgG levels also varied after malaria treatment, with significantly higher anti-ORF59 at day 0 but elevated ORF38, ORF73, and K8.1 at day 3. Acute malaria episodes is characterized by perturbation of KSHV latency in seropositive children, providing further evidence that malaria endemicity contributes to the observed increase in endemic KS incidence in sub-Saharan Africa.

Autophagy regulates long-term cross-presentation by murine dendritic cells.

Autophagy has been reported to be involved in supporting antigen cross-presentation by dendritic cells (DCs). We have shown that DCs have the ability to store antigen for a prolonged time in endolysosomal compartments and thereby sustain MHCI antigen cross-presentation to CD8+ T cells. In the current study, we investigated the role of autophagy in long-term antigen presentation. We show that the autophagy machinery has a negative impact on storage of antigen in DCs. Atg5-/- DCs which are deficient in autophagy or DCs treated with common autophagy inhibitors showed enhanced antigen storage and antigen cross-presentation. This augmented antigen cross-presentation effect is independent of altered proteasome enzyme activity or MHCI surface expression on DCs. We visualized that the storage compartments are in close proximity to LC3 positive autophagosomes. Our results indicate that autophagosomes disrupt antigen storage in DCs and thereby regulate long-term MHCI cross-presentation.

T-cell memory in tissues.

Immunological memory equips our immune system to respond faster and more effectively against reinfections. This acquired immunity was originally attributed to long-lived, memory T and B cells with body wide access to peripheral and secondary lymphoid tissues. In recent years, it has been realized that both innate and adaptive immunity to a large degree depends on resident immune cells that act locally in barrier tissues including tissue-resident memory T cells (Trm). Here, we will discuss the phenotype of these Trm in mice and humans, the tissues and niches that support them, and their function, plasticity, and transcriptional control. Their unique properties enable Trm to achieve long-lived immunological memory that can be deposited in nearly every organ in response to acute and persistent infection, and in response to cancer. However, Trm may also induce substantial immunopathology in allergic and autoimmune disease if their actions remain unchecked. Therefore, inhibitory and activating stimuli appear to balance the actions of Trm to ensure rapid proinflammatory responses upon infection and to prevent damage to host tissues under steady state conditions.

Attenuated immune control of Epstein-Barr virus in humanized mice is associated with the multiple sclerosis risk factor HLA-DR15.

Immune responses to Epstein-Barr virus (EBV) infection synergize with the main genetic risk factor HLA-DRB1*15:01 (HLA-DR15) to increase the likelihood to develop the autoimmune disease multiple sclerosis (MS) at least sevenfold. In order to gain insights into this synergy, we investigated HLA-DR15 positive human immune compartments after reconstitution in immune-compromised mice (humanized mice) with and without EBV infection. We detected elevated activation of both CD4+ and CD8+ T cells in HLA-DR15 donor-reconstituted humanized mice at steady state, even when compared to immune compartments carrying HLA-DRB1*04:01 (HLA-DR4), which is associated with other autoimmune diseases. Increased CD8+ T cell expansion and activation was also observed in HLA-DR15 donor-reconstituted humanized mice after EBV infection. Despite this higher immune activation, EBV viral loads were less well controlled in the context of HLA-DR15. Indeed, HLA-DR15-restricted CD4+ T cell clones recognized EBV-transformed B cell lines less efficiently and demonstrated cross-reactivity toward allogeneic target cells and one MS autoantigen. These findings suggest that EBV as one of the main environmental risk factors and HLA-DR15 as the main genetic risk factor for MS synergize by priming hyperreactive T-cell compartments, which then control the viral infection less efficiently and contain cross-reactive CD4+ T cell clones.

Reduced frequency of cytotoxic CD56dim CD16+ NK cells leads to impaired antibody-dependent degranulation in EBV-positive classical Hodgkin lymphoma.

Around 30-50% of classical Hodgkin lymphoma (cHL) cases in immunocompetent individuals from industrialized countries are associated with the B-lymphotropic Epstein-Barr virus (EBV). Although natural killer (NK) cells exhibit anti-viral and anti-tumoral functions, virtually nothing is known about quantitative and qualitative differences in NK cells in patients with EBV+ cHL vs. EBV- cHL. Here, we prospectively investigated 36 cHL patients without known immune suppression or overt immunodeficiency at diagnosis. All 10 EBV+ cHL patients and 25 out 26 EBV- cHL were seropositive for EBV antibodies, and EBV+ cHL patients presented with higher plasma EBV DNA levels compared to EBV- cHL patients. We show that the CD56dim CD16+ NK cell subset was decreased in frequency in EBV+ cHL patients compared to EBV- cHL patients. This quantitative deficiency translates into an impaired CD56dim NK cell mediated degranulation toward rituximab-coated HLA class 1 negative lymphoblastoid cells in EBV+ compared to EBV- cHL patients. We finally observed a trend to a decrease in the rituximab-associated degranulation and ADCC of in vitro expanded NK cells of EBV+ cHL compared to healthy controls. Our findings may impact on the design of adjunctive treatment targeting antibody-dependent cellular cytotoxicity in EBV+ cHL.

Correction: Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice.

[This corrects the article DOI: 10.1371/journal.ppat.1008477.].

Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice.

Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication after organ transplantation frequently associated with the Epstein-Barr virus (EBV). Immunosuppressive treatment is thought to allow the expansion of EBV-infected B cells, which often express all eight oncogenic EBV latent proteins. Here, we assessed whether HLA-A2 transgenic humanized NSG mice treated with the immunosuppressant FK506 could be used to model EBV-PTLD. We found that FK506 treatment of EBV-infected mice led to an elevated viral burden, more frequent tumor formation and diminished EBV-induced T cell responses, indicative of reduced EBV-specific immune control. EBV latency III and lymphoproliferation-associated cellular transcripts were up-regulated in B cells from immunosuppressed animals, akin to the viral and host gene expression pattern found in EBV-PTLD. Utilizing an unbiased gene expression profiling approach, we identified genes differentially expressed in B cells of EBV-infected animals with and without FK506 treatment. Upon investigating the most promising candidates, we validated sCD30 as a marker of uncontrolled EBV proliferation in both humanized mice and in pediatric patients with EBV-PTLD. High levels of sCD30 have been previously associated with EBV-PTLD in patients. As such, we believe that humanized mice can indeed model aspects of EBV-PTLD development and may prove useful for the safety assessment of immunomodulatory therapies.