Multiple sclerosis (MS) is an autoimmune disorder where T cells attack neurons in the central nervous system (CNS) leading to demyelination and neurological deficits. A driver of increased MS risk is ...the soluble form of the interleukin-7 receptor alpha chain gene (sIL7R) produced by alternative splicing of IL7R exon 6. Here, we identified the RNA helicase DDX39B as a potent activator of this exon and consequently a repressor of sIL7R, and we found strong genetic association of DDX39B with MS risk. Indeed, we showed that a genetic variant in the 5′ UTR of DDX39B reduces translation of DDX39B mRNAs and increases MS risk. Importantly, this DDX39B variant showed strong genetic and functional epistasis with allelic variants in IL7R exon 6. This study establishes the occurrence of biological epistasis in humans and provides mechanistic insight into the regulation of IL7R exon 6 splicing and its impact on MS risk.
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•DDX39B is a potent activator of IL7R exon 6 splicing and a repressor of sIL7R•DDX39B genetic variants are significantly associated with MS risk•The 5′ UTR DDX39B variant reduces protein levels by decreasing translation efficiency•This variant shows strong genetic and functional epistasis with IL7R rs6897932
Two genes interact epistatically in multiple sclerosis risk in humans.
Broadly neutralizing antibodies (bNAbs), known to mediate immune control of HIV-1 infection, only develop in a small subset of HIV-1 infected individuals. Despite being traditionally associated with ...patients with high viral loads, bNAbs have also been observed in therapy naïve HIV-1+ patients naturally controlling virus replication Virus Controllers (VCs). Thus, dissecting the bNAb response in VCs will provide key information about what constitutes an effective humoral response to natural HIV-1 infection. In this study, we identified a polyclonal bNAb response to natural HIV-1 infection targeting CD4 binding site (CD4bs), V3-glycan, gp120-gp41 interface and membrane-proximal external region (MPER) epitopes on the HIV-1 envelope (Env). The polyclonal antiviral antibody (Ab) response also included antibody-dependent cellular phagocytosis of clade AE, B and C viruses, consistent with both the Fv and Fc domain contributing to function. Sequence analysis of
from one of the VCs revealed features consistent with potential immune pressure and virus escape from V3-glycan targeting bNAbs. Epitope mapping of the polyclonal bNAb response in VCs with bNAb activity highlighted the presence of gp120-gp41 interface and CD4bs antibody classes with similar binding profiles to known potent bNAbs. Thus, these findings reveal the induction of a broad and polyfunctional humoral response in VCs in response to natural HIV-1 infection.
Both neutralization and antibody-dependent cellular cytotoxicity (ADCC) may be required for effective protection against HIV-1 infection. While there is extensive information on the targets of early ...neutralizing antibody (nAb) responses, much less is known about the targets of ADCC responses, which are more difficult to characterize. In four individuals recruited during acute HIV-infection, ADCC responses were detected 3-7 weeks prior to nAb responses. To determine the relative influence of ADCC and nAb responses on virus evolution, we performed an in-depth investigation of one individual (CAP63) who showed the highest nAb and ADCC responses. Both nAbs and ADCC antibodies targeted the V4 region of the Env, although there were some differences in epitope recognition. We identified accelerated viral evolution in this region concurrent with emergence of nAb activity, but not ADCC activity. Deep sequencing demonstrated that most nAb escape mutations were strongly selected for, however one nAb escape mutation that rendered the virus highly susceptible to autologous ADCC responses, was suppressed despite not affecting viral fitness. This escape mutation also rendered the virus more sensitive to autologous responses, as well as monoclonal antibodies targeting CD4-induced epitopes, compared to the wildtype virus. In conclusion, ADCC responses and nAbs in donor CAP63 recognized overlapping but unique epitopes in the V4 region, and while ADCC activity was present prior to nAbs, it did not drive viral evolution during this time. However, ADCC responses may select against nAb escape pathways that expose other common ADCC epitopes thereby restricting viral replication and expansion.
A paradigm shifting study demonstrated that induction of MHC class E and II-restricted CD8+ T cells was associated with the clearance of SIV infection in rhesus macaques. Another recent study ...highlighted the presence of HIV-1-specific class II-restricted CD8+ T cells in HIV-1 patients who naturally control infection (virus controllers; VCs). However, questions regarding class II-restricted CD8+ T cells ontogeny, distribution across different HIV-1 disease states and their role in viral control remain unclear. In this study, we investigated the distribution and anti-viral properties of HLA-DRB1*0701 and DQB1*0501 class II-restricted CD8+ T cells in different HIV-1 patient cohorts; and whether class II-restricted CD8+ T cells represent a unique T cell subset. We show that memory class II-restricted CD8+ T cell responses were more often detectable in VCs than in chronically infected patients, but not in healthy seronegative donors. We also demonstrate that VC CD8+ T cells inhibit virus replication in both a class I- and class II-dependent manner, and that in two VC patients the class II-restricted CD8+ T cells with an anti-viral gene signature expressed both CD4+ and CD8+ T cell lineage-specific genes. These data demonstrated that anti-viral memory class II-restricted CD8+ T cells with hybrid CD4+ and CD8+ features are present during natural HIV-1 infection.
Type 5 human adenoviruses (Ad5) deleted of genes encoding the early region 1B 55-kDa (E1B55K) protein including Onyx-015 (dl1520) and H101 are best known for their oncolytic potential. As a vaccine ...vector the E1B55K deletion may allow for the insertion of a transgene nearly 1,000 base pairs larger than now possible. This has the potential of extending the application for which the vectors are clinically known. However, the immune priming ability of E1B55K-deleted vectors is unknown, undermining our ability to gauge their usefulness in vaccine applications. For this reason, we created an E1B55K-deleted Ad5 vector expressing full-length single chain HIVBaLgp120 attached to a flexible linker and the first two domains of rhesus CD4 (rhFLSC) in exchange for the E3 region. In cell-based experiments the E1B55K-deleted vector promoted higher levels of innate immune signals including chemokines, cytokines, and the NKG2D ligands MIC A/B compared to an E1B55K wild-type vector expressing the same immunogen. Based on these results we evaluated the immune priming ability of the E1B55K-deleted vector in mice. The E1B55K-deleted vector promoted similar levels of Ad5-, HIVgp120, and rhFLSC-specific cellular and humoral immune responses as the E1B55K wild-type vector. In pre-clinical HIV-vaccine studies the wild-type vector has been employed as part of a very effective prime-boost strategy. This study demonstrates that E1B55K-deleted adenoviruses may serve as effective vaccine delivery vectors.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Diversity in specificity of polyclonal antibody (pAb) responses is extensively investigated in vaccine efficacy or immunological evaluations, but the heterogeneity in antibody avidity is rarely ...probed as convenient tools are lacking. Here we have developed a polyclonal antibodies avidity resolution tool (PAART) for use with label-free techniques, such as surface plasmon resonance and biolayer interferometry, that can monitor pAb-antigen interactions in real time to measure dissociation rate constant (
) for defining avidity. PAART utilizes a sum of exponentials model to fit the dissociation time-courses of pAb-antigens interactions and resolve multiple
contributing to the overall dissociation. Each
value of pAb dissociation resolved by PAART corresponds to a group of antibodies with similar avidity. PAART is designed to identify the minimum number of exponentials required to explain the dissociation course and guards against overfitting of data by parsimony selection of best model using Akaike information criterion. Validation of PAART was performed using binary mixtures of monoclonal antibodies of same specificity but differing in
of the interaction with their epitope. We applied PAART to examine the heterogeneity in avidities of pAb from malaria and typhoid vaccinees, and individuals living with HIV-1 that naturally control the viral load. In many cases, two to three
were dissected indicating the heterogeneity of pAb avidities. We showcase examples of affinity maturation of vaccine induced pAb responses at component level and enhanced resolution of heterogeneity in avidity when antigen-binding fragments (Fab) are used instead of polyclonal IgG antibodies. The utility of PAART can be manifold in examining circulating pAb characteristics and could inform vaccine strategies aimed to guide the host humoral immune response.
We have recently shown that MUC16, a component of the glycocalyx of some mucosal barriers, has elevated binding to the G0 glycoform of the Fc portion of IgG. Therefore, IgG from patients chronically ...infected with human immunodeficiency virus (HIV), who typically exhibit increased amounts of G0 glycoforms, showed increased MUC16 binding compared to uninfected controls. Using the rhesus macaque simian immunodeficiency virus SIVmac251 model, we can compare plasma antibodies before and after chronic infection. We find increased binding of IgG to MUC16 after chronic SIV infection. Antibodies isolated for tight association with MUC16 (MUC16-eluted antibodies) show reduced FcγR engagement and antibody-dependent cellular cytotoxicity (ADCC) activity. The glycosylation profile of these IgGs was consistent with a decrease in FcγR engagement and subsequent ADCC effector function, as they contain a decrease in afucosylated bisecting glycoforms that preferentially bind FcγRs. Testing of the SIV antigen specificity of IgG from SIV-infected macaques revealed that the MUC16-eluted antibodies were enriched for certain specific epitopes, including regions of gp41 and gp120. This enrichment of specific antigen responses for fucosylated bisecting glycoforms and the subsequent association with MUC16 suggests that the immune response has the potential to direct specific epitope responses to localize to the glycocalyx through interaction with this specific mucin.
Understanding how antibodies are distributed in the mucosal environment is valuable for developing a vaccine to block HIV infection. Here, we study an IgG binding activity in MUC16, potentially representing a new IgG effector function that would concentrate certain antibodies within the glycocalyx to trap pathogens before they can reach the underlying columnar epithelial barriers. These studies reveal that rhesus macaque IgG responses during chronic SIV infection generate increased antibodies that bind MUC16, and interestingly, these MUC16-tethered antibodies are enriched for binding to certain antigens. Therefore, it may be possible to direct HIV vaccine-generated responses to associate with MUC16 and enhance the antibody's ability to mediate immune exclusion by trapping virions within the glycocalyx and preventing the virus from reaching immune target cells within the mucosa. This concept will ultimately have to be tested in the rhesus macaque model, which is shown here to have MUC16-targeted antigen responses.
There is a lack of a clear understanding of what constitutes a potent natural immune response capable of mediating post-infection protection in an HIV-1 infection setting. Thus, the goal of my ...dissertation was to dissect the immune responses to HIV-1 infection associated with protection using a model for natural control from HIV-1 infection. The first objective was to expand our understanding of what constitutes an effective CD8+ T cell response, while the second objective was to bridge Fab and Fc-mediated antibody effector functions associated with natural control of HIV-1 infection. The conventional T cell paradigm dictates CD8+ T cell recognition of peptides in the context of MHC class I. Remarkably, a paradigm shifting study demonstrated that induction of MHC class E and II-restricted CD8+ T cells was associated with the clearance of SIV infection in rhesus macaques. However, the induction of the unconventional class II-restricted CD8+ T cells in a natural HIV-1 infection setting, their ontogeny, distribution across different HIV-1 disease states and their role in viral control remain unclear. In the first part of my dissertation research, I hypothesized that HIV-1 specific class II-restricted CD8+ T cells are present in patients naturally controlling HIV-1 infection (Virus Controllers). I also hypothesized that HLA class II-restricted CD8+ T cells capable of suppressing viral replication in autologous CD4+ T cells are ‘ex-CD4+ T cells’ with CD8-like anti-viral effector functions. I not only showed that HIV-1 specific HLA class II-restricted CD8+ T cells are present in HIV-1 VCs, but that memory class II-restricted CD8+ T cell responses were more often detectable in VCs than in chronic viremics, and absent in healthy donors. I also demonstrated that VC CD8+ T cells inhibit virus replication in both a class I- and II-dependent manner and that in two VC patients the class II-restricted CD8+ T cells with an anti-viral gene signature expressed both CD4+ and CD8+ T cell lineage-specific genes. These data demonstrated that anti-viral memory class II-restricted CD8+ T cells with hybrid CD4+ and CD8+ features are present during natural HIV-1 infection. The presence of these atypical CD8+ T cells during HIV-1 infection redefines what may constitute an effective CD8+ T cell response. Moreover, my findings provide a potentially effective target for CD8+ T cell vaccine strategies given that these cells are readily detectable at a higher frequency in HIV+ patients spontaneously controlling virus replication compared to chronic patients. The second objective of my dissertation was to bridge the Fab-mediated neutralization and Fc-mediated humoral effector functions associated with natural control of HIV-1 infection by determining whether neutralization breadth was coordinated with the Fc effector function, antibody-dependent cellular phagocytosis (ADCP), in HIV-1 VCs. The Fc domain of antibodies enhances the in vivo neutralization potencies of broadly neutralization antibodies while the Fc effector functions antibody-dependent complement deposition (ADCD) and antibody-dependent cellular trogocytosis (ADCT) have been associated with the development of neutralization breadth. However, the potential coordination of ADCP, an Fc effector function associated with decreased infection risk and also enriched in HIV-1 VCs, with neutralization breadth and how such a relationship might mediate enhanced virus control remain unclear. In the second part of my dissertation research, I hypothesized that the development of nAb breadth was coordinated with ADCP breadth in HIV-1 VCs. I also hypothesized that the HIV-1 VC broadly neutralizing antibody (bNAb) polyclonal response targeted both known bNAb epitopes and previously unidentified vulnerable epitopes on the HIV-1 Envelope. By utilizing a novel polyclonal epitope mapping approach, I also hypothesized that the polyclonal antibody responses (neutralizing and non-neutralizing) to natural HIV-1 infection can be mapped by electron microscopy. I discovered a coordination between the nAb breadth and virion ADCP breadth in a subset of HIV-1 VCs characterized by the targeting of the CD4bs, V3 glycan and MPER known bNAb epitopes on the HIV-1 envelope that potentially act in synergy to mediate the broad nAb and ADCP responses observed in this patient cohort. The HIV-1 VCs with bNAb activity contain a potent class of CD4bs bNAbs that has never been reported in HIV-1 VCs without autoimmunity before. Thus, these findings not only suggest the presence of potent humoral responses in HIV-1 VCs, but also suggest a possible role of ADCP in modulating the bNAb response in a low viremia setting. Taken together, these findings highlight the presence of novel immune responses and multi-compartment associations in a subset of patients mediating natural control of viral infection and hence indicating the need to further investigate and elucidate the mechanistic processes behind these immune responses and relationships. This body of work sheds light on the complex anti-viral immune response in HIV-1 VCs and provides potential novel targets for future HIV vaccine design.
Abstract
CD4+ and CD8+ T cells are conventionally considered as separate lineages characterized by distinct surface markers, function and MHC restriction. However, CD4+ T cells have a ...recently-appreciated capacity to acquire CD8+ T cell-like characteristics upon TCR-mediated activation. We have identified a novel, CD4+ T cell-derived effector population in mice that is CD4−CD8αβ+ and MHCII-restricted. These cells express a unique lineage-intermediate transcriptional program (ThPOKlo-int Runx3hi-int), upregulate granzyme B/IFN-γ expression and exhibit antigen-specific cytotoxicity. Following acute infection with LCMV Armstrong in wild-type mice or with Listeria monocytogenes in MHCII-restricted, TCR-transgenic mice, we have used CD4+ T cell lineage tracing, MHCII tetramer staining and TCR repertoire sequencing techniques to demonstrate that ex-CD4+ T cell responses can be induced in vivo and originate from bona fide CD4+ T cells. We also report ex-CD4+-like T cells in human HIV patients that are CD8+ and exhibit MHCII-blockable antiviral activity. This population is diminished in elite controller patients upon loss of virus control, indicating that it may contribute to protection against HIV. Finally, we have generated preliminary evidence that ex-CD4+ T cell generation is controlled by an autophagy-related mechanism. CD4+ T cell-specific deficiency in either of the autophagy regulators Vps34 or Atg7 results in dramatic loss of CD4 expression upon antigen stimulation, concomitant with skewing towards the CD8+ transcriptional program in Vps34-deficient cells. We propose that the ex-CD4+ T cell lineage dramatically extends the paradigm of αβ T cell lineage plasticity and may indicate new antiviral vaccine strategies.
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