Comprehensive Cross-Clade Characterization of Antibody-Mediated Recognition, Complement-Mediated Lysis, and Cell-Mediated Cytotoxicity of HIV-1 Envelope-Specific Antibodies toward Eradication of the HIV-1 Reservoir.

Immunotherapy with passive administration of broadly neutralizing HIV-1 envelope-specific antibodies (bnAbs) in the setting of established infection in vivo has yielded mixed results. The contribution of different antibodies toward the direct elimination of infected cells is poorly understood. In this study, we determined the ability of 12 well-characterized anti-HIV-1 neutralizing antibodies to recognize and eliminate primary CD4 T cells infected with HIV-1 belonging to clades A, B, C, and D, via antibody-dependent complement-mediated lysis (ADCML) and antibody-dependent cell-mediated cytotoxicity (ADCC), in vitro We further tested unique combinations of these antibodies to determine the optimal antibody cocktails to be tested in future clinical trials. We report that antibody binding to infected CD4 T cells is highly variable and correlates with ADCML and ADCC processes. Particularly, antibodies targeting the envelope glycan shield (2G12) and V1/V2 site (PG9, PG16, and PGT145) are best at recognizing HIV-1-infected CD4 T cells. However, only PG9 and PG16 and their combinations with other bnAbs sufficiently induced the elimination of HIV-1-infected CD4 T cells by ADCML, ADCC, or both. Notably, CD4 binding site antibodies VRC01, 3BNC117, and NIH45-46 G54W did not exhibit recognition of infected cells and were unable to induce their killing. Future trials geared toward the development of a cure for HIV/AIDS should incorporate V1/V2 antibodies for maximal clearance of infected cells. With the use of only primary immune cells, we conducted a comprehensive cross-clade physiological analysis to aid the direction of antibodies as therapeutics toward the development of a cure for HIV/AIDS.IMPORTANCE Several antibodies capable of neutralizing the majority of circulating HIV-1 strains have been identified to date and have been shown to prevent infection in animal models. However, the use of combinations of such broadly neutralizing antibodies (bnAbs) for the treatment and eradication of HIV-1 in infected humans remains uncertain. In this study, we tested the ability of bnAbs to directly recognize and eliminate primary human CD4 T cells infected with diverse HIV-1 strains representative of the global epidemic by antibody-dependent pathways. We also tested several combinations of bnAbs in our assays in order to maximize the clearance of infected cells. We show that the ability of bnAbs to identify and kill infected cells is highly variable and that only a few of them are able to exert this function. Our data will help guide the formulation of bnAbs to test in future human trials aimed at the development of a cure.

Tim-3 is a Marker of Plasmacytoid Dendritic Cell Dysfunction during HIV Infection and Is Associated with the Recruitment of IRF7 and p85 into Lysosomes and with the Submembrane Displacement of TLR9.

In chronic diseases, such as HIV infection, plasmacytoid dendritic cells (pDCs) are rendered dysfunctional, as measured by their decreased capacity to produce IFN-α. In this study, we identified elevated levels of T cell Ig and mucin-domain containing molecule-3 (Tim-3)-expressing pDCs in the blood of HIV-infected donors. The frequency of Tim-3-expressing pDCs correlated inversely with CD4 T cell counts and positively with HIV viral loads. A lower frequency of pDCs expressing Tim-3 produced IFN-α or TNF-α in response to the TLR7 agonists imiquimod and Sendai virus and to the TLR9 agonist CpG. Thus, Tim-3 may serve as a biomarker of pDC dysfunction in HIV infection. The source and function of Tim-3 was investigated on enriched pDC populations from donors not infected with HIV. Tim-3 induction was achieved in response to viral and artificial stimuli, as well as exogenous IFN-α, and was PI3K dependent. Potent pDC-activating stimuli, such as CpG, imiquimod, and Sendai virus, induced the most Tim-3 expression and subsequent dysfunction. Small interfering RNA knockdown of Tim-3 increased IFN-α secretion in response to activation. Intracellular Tim-3, as measured by confocal microscopy, was dispersed throughout the cytoplasm prior to activation. Postactivation, Tim-3 accumulated at the plasma membrane and associated with disrupted TLR9 at the submembrane. Tim-3-expressing pDCs had reduced IRF7 levels. Furthermore, intracellular Tim-3 colocalized with p85 and IRF7 within LAMP1+ lysosomes, suggestive of a role in degradation. We conclude that Tim-3 is a biomarker of dysfunctional pDCs and may negatively regulate IFN-α, possibly through interference with TLR signaling and recruitment of IRF7 and p85 into lysosomes, enhancing their degradation.

HIV-Specific Granzyme B-Secreting but Not Gamma Interferon-Secreting T Cells Are Associated with Reduced Viral Reservoirs in Early HIV Infection.

A major barrier to a human immunodeficiency virus type 1 (HIV-1) infection cure is the establishment of a viral reservoir in spite of combined antiretroviral therapy (cART). It is unclear how HIV-specific cytotoxic T lymphocytes (CTLs) influence the size of the reservoir in early HIV infection. Twenty-eight subjects with early HIV infection were recruited to receive cART and followed for 48 weeks. HIV reservoirs in peripheral CD4+ T cells measured by cell-associated proviral DNA and viral outgrowth cultures were determined at baseline and after 48 weeks of cART. At baseline, granzyme B and gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assays were performed with peptides spanning the HIV proteome. All subjects had detectable HIV-specific granzyme B and IFN-γ responses at baseline. The quantity and specificity of granzyme B responses did not correlate with IFN-γ responses. For granzyme B, Tat/Rev was the most dominant whereas for IFN-γ, Gag predominated. HIV-specific granzyme B T cell responses negatively correlated with HIV proviral loads at baseline and at 48 weeks and with replication-competent viral infectious units per million (IUPM) CD4+ T cells at baseline but not significantly at 48 weeks. Tat/Rev-, Env-, Gag-, and Vif-specific granzyme B responses correlated most strongly with reservoir control. There was no correlation of HIV-specific IFN-γ responses with reservoir size at baseline or at 48 weeks. The majority of granzyme B responses were contributed by CD8+ T cells. Thus, our findings suggest that the induction of potent granzyme B-producing CTLs to Tat, Rev, Env, Gag, and Vif during early infection may be able to prevent the establishment of a large viral reservoir, thereby facilitating a reduced HIV burden.IMPORTANCE A major barrier to the cure of human immunodeficiency virus type 1 (HIV-1) infection is the establishment of a viral reservoir that must be significantly reduced or eradicated entirely to enable a cure. Combined antiretroviral therapy (cART) alone is unable to clear this viral reservoir. It has been shown that CD8+ cytotoxic T lymphocytes (CTLs) are important in controlling early HIV infection by reducing plasma viremia. However, it is not known if these HIV-specific CTLs influence the establishment of the viral reservoir in early HIV infection. We show that HIV-specific granzyme B responses targeting HIV Tat/Rev, Env, Gag, and Vif, but not IFN-γ responses, are associated with reduced virus reservoirs at baseline and at 48 weeks of cART. These findings shed light on the nature of the effector CTL response that might limit reservoir size with implications for cure research and HIV vaccines.

HCV Specific IL-21 Producing T Cells but Not IL-17A Producing T Cells Are Associated with HCV Viral Control in HIV/HCV Coinfection.

BACKGROUND: Decreased hepatitis C virus (HCV) clearance, faster cirrhosis progression and higher HCV RNA levels are associated with Human Immunodeficiency virus (HIV) coinfection. The CD4+ T helper cytokines interleukin (IL)-21 and IL-17A are associated with virus control and inflammation, respectively, both important in HCV and HIV disease progression. Here, we examined how antigen-specific production of these cytokines during HCV mono and HIV/HCV coinfection was associated with HCV virus control. METHODS: We measured HCV-specific IL-21 and IL-17A production by transwell cytokine secretion assay in PBMCs from monoinfected and coinfected individuals. Viral control was determined by plasma HCV RNA levels. RESULTS: In acutely infected individuals, those able to establish transient/complete HCV viral control tended to have stronger HCV-specific IL-21-production than non-controllers. HCV-specific IL-21 production also correlated with HCV viral decline in acute infection. Significantly stronger HCV-specific IL-21 production was detected in HAART-treated coinfected individuals. HCV-specific IL-17A production was not associated with lower plasma HCV RNA levels in acute or chronic HCV infection and responses were stronger in HIV coinfection. HCV-specific IL-21/ IL-17A responses did not correlate with microbial translocation or fibrosis. Exogenous IL-21 treatment of HCV-specific CD8+ T cells from monoinfected individuals enhanced their function although CD8+ T cells from coinfected individuals were somewhat refractory to the effects of IL-21. CONCLUSIONS: These data show that HCV-specific IL-21 and IL-17A-producing T cells are induced in HIV/HCV coinfection. In early HIV/HCV coinfection, IL-21 may contribute to viral control, and may represent a novel tool to enhance acute HCV clearance in HIV/HCV coinfected individuals.

Molecular requirements for MHC class II alpha-chain engagement and allelic discrimination by the bacterial superantigen streptococcal pyrogenic exotoxin C.

Superantigens (SAgs) are microbial toxins that bind to both TCR beta-chain variable domains (Vbetas) and MHC class II molecules, resulting in the activation of T cells in a Vbeta-specific manner. It is now well established that different isoforms of MHC II molecules can play a significant role in the immune response to bacterial SAgs. In this work, using directed mutational studies in conjunction with functional analyses, we provide a complete functional map of the low-affinity MHC II alpha-chain binding interface of the SAg streptococcal pyrogenic exotoxin C (SpeC) and identify a functional epitope in the beta-barrel domain that is required for the activation of T cells. Using cell lines that exclusively express individual MHC II isoforms, our studies provide a molecular basis for the selectivity of SpeC-MHC II recognition, and provide one mechanism by how SAgs are capable of distinguishing between different MHC II alleles.

Neutralization of multiple staphylococcal superantigens by a single-chain protein consisting of affinity-matured, variable domain repeats.

Staphylococcus aureus secretes various toxins that act as superantigens by stimulating a large fraction of the host's T cells. Toxin binding to variable domains of T cell receptor beta chains (Vbeta) leads to massive release of inflammatory molecules and potentially to toxic shock syndrome (TSS). Previously, we generated soluble forms of different Vbeta domains with a high affinity for binding superantigens. However, a broader spectrum antagonist is required for the neutralization of multiple toxins. In the present study, we expressed Vbeta domains in tandem as a single-chain protein and neutralized the clinically important superantigens staphylococcal enterotoxin B and TSS toxin-1 with a single agent.

A novel loop domain in superantigens extends their T cell receptor recognition site.

Superantigens (SAGs) interact with host immune receptors to induce a massive release of inflammatory cytokines that can lead to toxic shock syndrome and death. Bacterial SAGs can be classified into five distinct evolutionary groups. Group V SAGs are characterized by the alpha3-beta8 loop, a unique approximately 15 amino acid residue extension that is required for optimal T cell activation. Here, we report the X-ray crystal structures of the group V SAG staphylococcal enterotoxin K (SEK) alone and in complex with the TCR hVbeta5.1 domain. SEK adopts a unique TCR binding orientation relative to other SAG-TCR complexes, which results in the alpha3-beta8 loop contacting the apical loop of framework region 4, thereby extending the known TCR recognition site of SAGs. These interactions are absolutely required for TCR binding and T cell activation by SEK, and dictate the TCR Vbeta domain specificity of SEK and other group V SAGs.

Crystal structure of the streptococcal superantigen SpeI and functional role of a novel loop domain in T cell activation by group V superantigens.

Superantigens (SAgs) are potent microbial toxins that bind simultaneously to T cell receptors (TCRs) and class II major histocompatibility complex molecules, resulting in the activation and expansion of large T cell subsets and the onset of numerous human diseases. Within the bacterial SAg family, streptococcal pyrogenic exotoxin I (SpeI) has been classified as belonging to the group V SAg subclass, which are characterized by a unique, relatively conserved approximately 15 amino acid extension (amino acid residues 154 to 170 in SpeI; herein referred to as the alpha3-beta8 loop), absent in SAg groups I through IV. Here, we report the crystal structure of SpeI at 1.56 A resolution. Although the alpha3-beta8 loop in SpeI is several residues shorter than that of another group V SAg, staphylococcal enterotoxin serotype I, the C-terminal portions of these loops, which are located adjacent to the putative TCR binding site, are structurally similar. Mutagenesis and subsequent functional analysis of SpeI indicates that TCR beta-chains are likely engaged in a similar general orientation as other characterized SAgs. We show, however, that the alpha3-beta8 loop length, and the presence of key glycine residues, are necessary for optimal activation of T cells. Based on Vbeta-skewing analysis of human T cells activated with SpeI and structural models, we propose that the alpha3-beta8 loop is positioned to form productive intermolecular contacts with the TCR beta-chain, likely in framework region 3, and that these contacts are required for optimal TCR recognition by SpeI, and likely all other group V SAgs.

Molecular basis of TCR selectivity, cross-reactivity, and allelic discrimination by a bacterial superantigen: integrative functional and energetic mapping of the SpeC-Vbeta2.1 molecular interface.

Superantigens activate large fractions of T cells through unconventional interactions with both TCR beta-chain V domains (Vbetas) and MHC class II molecules. The bacterial superantigen streptococcal pyrogenic exotoxin C (SpeC) primarily stimulates human Vbeta2(+) T cells. Herein, we have analyzed the SpeC-Vbeta2.1 interaction by mutating all SpeC residues that make contact with Vbeta2.1 and have determined the energetic and functional consequences of these mutations. Our comprehensive approach, including mutagenesis, functional readouts from both bulk T cell populations, and an engineered Vbeta2.1(+) Jurkat T cell, as well as surface plasmon resonance binding analysis, has defined the SpeC "functional epitope" for TCR engagement. Although only two SpeC residues (Tyr(15) and Arg(181)) are critical for activation of virtually all human CD3(+) T cells, a larger cluster of four hot spot residues are required for interaction with Vbeta2.1. Three of these residues (Tyr(15), Phe(75), and Arg(181)) concentrate their binding energy on the CDR2 loop residue Ser(52a), a noncanonical residue insertion found only in Vbeta2 and Vbeta4 chains. Plasticity of this loop is important for recognition by SpeC. Although SpeC interacts with the Vbeta2.1 hypervariable CDR3 loop, our data indicate these contacts have little to no influence on the functional interaction with Vbeta2.1. These studies also provide a molecular basis for selectivity and cross-reactivity of SpeC-TCR recognition and reveal a degree of fine specificity in these interactions, whereby certain SpeC mutants are capable of distinguishing between different alleles of the same Vbeta domain subfamily.

Bacterial superantigens bypass Lck-dependent T cell receptor signaling by activating a Galpha11-dependent, PLC-beta-mediated pathway.

The paradigm to explain antigen-dependent T cell receptor (TCR) signaling is based on the activation of the CD4 or CD8 coreceptor-associated kinase Lck. It is widely assumed that this paradigm is also applicable to signaling by bacterial superantigens. However, these bacterial toxins can activate human T cells lacking Lck, suggesting the existence of an additional pathway of TCR signaling. Here we showed that this alternative pathway operates in the absence of Lck-dependent tyrosine-phosphorylation events and was initiated by the TCR-dependent activation of raft-enriched heterotrimeric Galpha11 proteins. This event, in turn, activated a phospholipase C-beta and protein kinase C-mediated cascade that turned on the mitogen-activated protein kinases ERK-1 and ERK-2, triggered Ca(2+) influx, and translocated the transcription factors NF-AT and NF-kappaB to the nucleus, ultimately inducing the production of interleukin-2 in Lck-deficient T cells. The triggering of this alternative pathway by superantigens suggests that these toxins use a G protein-coupled receptor as a coreceptor on T cells.

Long-range cooperative binding effects in a T cell receptor variable domain.

Although cellular processes depend on protein-protein interactions, our understanding of molecular recognition between proteins remains far from comprehensive. Protein-protein interfaces are structural and energetic mosaics in which a subset of interfacial residues, called hot spots, contributes disproportionately to the affinity of the complex. These hot-spot residues can be further clustered into hot regions. It has been proposed that binding energetics between residues within a hot region are cooperative, whereas those between hot regions are strictly additive. If this idea held true for all protein-protein interactions, then energetically significant long-range conformational effects would be unlikely to occur. In the present study, we show cooperative binding energetics between distinct hot regions that are separated by >20 A. Using combinatorial mutagenesis and surface plasmon resonance binding analysis to dissect additivity and cooperativity in a complex formed between a variable domain of a T cell receptor and a bacterial superantigen, we find that combinations of mutations from each of two hot regions exhibited significant cooperative energetics. Their connecting sequence is composed primarily of a single beta-strand of the T cell receptor variable Ig domain, which has been observed to undergo a strand-switching event and does not form an integral part of the stabilizing core of this Ig domain. We propose that these cooperative effects are propagated through a dynamic structural network. Cooperativity between hot regions has significant implications for the prediction and inhibition of protein-protein interactions.