Evidence from C57BL/6 mice suggests that CD8+ T cells, specific to the immunodominant HSV-1 glycoprotein B (gB) H-2b–restricted epitope (gB498–505), protect against ocular herpes infection and disease. However, the possible role of CD8+ T cells, specific to HLA-restricted gB epitopes, in protective immunity seen in HSV-1–seropositive asymptomatic (ASYMP) healthy individuals (who have never had clinical herpes) remains to be determined. In this study, we used multiple prediction algorithms to identify 10 potential HLA-A*02:01–restricted CD8+ T cell epitopes from the HSV-1 gB amino acid sequence. Six of these epitopes exhibited high-affinity binding to HLA-A*02:01 molecules. In 10 sequentially studied HLA-A*02:01–positive, HSV-1–seropositive ASYMP individuals, the most frequent, robust, and polyfunctional CD8+ T cell responses, as assessed by a combination of tetramer, IFN-γ-ELISPOT, CFSE proliferation, CD107a/b cytotoxic degranulation, and multiplex cytokine assays, were directed mainly against epitopes gB342–350 and gB561–569. In contrast, in 10 HLA-A*02:01–positive, HSV-1–seropositive symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent clinical herpes disease) frequent, but less robust, CD8+ T cell responses were directed mainly against nonoverlapping epitopes (gB183–191 and gB441–449). ASYMP individuals had a significantly higher proportion of HSV-gB–specific CD8+ T cells expressing CD107a/b degranulation marker and producing effector cytokines IL-2, IFN-γ, and TNF-α than did SYMP individuals. Moreover, immunization of a novel herpes-susceptible HLA-A*02:01 transgenic mouse model with ASYMP epitopes, but not with SYMP epitopes, induced strong CD8+ T cell–dependent protective immunity against ocular herpes infection and disease. These findings should guide the development of a safe and effective T cell–based herpes vaccine.

Targeting the mucosal immune system of the genital tract (GT) with subunit vaccines failed to induce potent and durable local CD8+ T cell immunity, crucial for protection against many sexually transmitted viral (STV) pathogens, including herpes simplex virus type 2 (HSV-2) that causes genital herpes. In this study, we aimed to investigate the potential of a novel lipopeptide/adenovirus type 5 (Lipo/rAdv5) prime/boost mucosal vaccine for induction of CD8+ T cell immunity to protect the female genital tract from herpes. The lipopeptide and the rAdv5 vaccine express the immunodominant HSV-2 CD8+ T cell epitope (gB498-505) and both were delivered intravaginally (IVAG) in the progesterone-induced B6 mouse model of genital herpes. Compared to its homologous lipopeptide/lipopeptide (Lipo/Lipo); the Lipo/rAdv5 prime/boost immunized mice: (i) developed potent and sustained HSV-specific CD8+ T cells, detected in both the GT draining nodes (GT-DLN) and in the vaginal mucosa (VM); (ii) had significantly lower virus titers; (iii) had decreased overt signs of genital herpes disease; and (iv) did not succumb to lethal infection (p < 0.005), following intravaginal HSV-2 challenge. Polyfunctional CD8+ T cells, producing IFN-γ, TNF-α and IL-2 and exhibiting cytotoxic activity, were associated with protection (p < 0.005). The protective CD8+ T cell response was significantly compromised in the absence of the adaptor myeloid differentiation factor 88 (MyD88) (p = 0.0001). Taken together, these findings indicate that targeting the VM with a Lipo/rAdv5 prime/boost vaccine elicits a potent, MyD88-dependent, and long-lasting mucosal CD8+ T cell protective immunity against sexually transmitted herpes infection and disease.

The binding and activation of the discoidin domain receptor 1 by collagen has led to the conclusion that proteins from the extracellular matrix can directly induce receptor tyrosine kinase-mediated signaling cascades. A region in the extracellular domain of DDR1 homologous to the Dictyostelium discoideum protein discoidin-I is also present in the secreted human protein RS1. Mutations in RS1 cause retinoschisis, a genetic disorder characterized by ablation of the retina. By introducing point mutations into the discoidin domain of DDR1 at positions homologous to the retinoschisis mutations, ligand binding epitopes in the discoidin domain of DDR1 were mapped. Surprisingly, some residues only affected receptor phosphorylation, whereas others influenced both collagen-binding and receptor activation. Furthermore, two truncated DDR1 variants, lacking either the discoidin domain or the stalk region between the discoidin and transmembrane domain, were generated. We showed that (i) the discoidin domain was necessary and sufficient for collagen binding, (ii) only the region between discoidin and transmembrane domain was glycosylated, and (iii) the entire extracellular domain was essential for transmembrane signaling. Using these results, we were able to predict key sites in the collagen-binding epitope of DDR1 and to suggest a potential mechanism of signaling.Discoidin domain receptors 1 and 2 (DDR1 and DDR2) 1 have been recognized as a distinct tyrosine kinase receptor subfamily because of structural and functional homologies. In their extracellular region, both receptors show a domain homologous to the Dictyostelium discoideum protein discoidin I. DDR1 and DDR2 are also functionally related by the observation that collagen acts as cognate ligand for both receptors. Whereas DDR1 activation is achieved by all collagens so far tested (types I-VI and VIII), DDR2 is only activated by fibrillar collagens, in particular by collagen type I and type III. In contrast to most other tyrosine kinase receptors, activation of DDRs can take several hours (1, 2).The cDNA coding for human DDR1 has been cloned from several tissues or carcinoma cells (3-7). Gene orthologs to human DDR1 have been identified in mice, rats, and Caenorhabditis elegans (8 -10). Expression of human DDR1 is predominantly seen in epithelial cells, particularly from kidney, lung, gastrointestinal tract, and brain, but also in corneal and dermal fibroblasts (7, 9, 11-13). DDR1 seems to be also involved in the differentiation of cerebellar granular neurons (14). Upregulated DDR1 expression has been reported from breast, ovarian, esophagus and brain tumors (5,(15)(16)(17)(18)(19). Human DDR1 is located on chromosome 6p21.3 in close proximity to HLA genes, which belong to the telomeric region (class I) of the major histocompatibility complex (20). The juxtamembrane regions in DDR1 and DDR2 are much longer than in other receptor tyrosine kinases (176 and 147 amino acids, respectively). Furthermore, the extracellular domain of DDR1 is shed by an unidentified protease, res...

We recently found that the herpes simplex virus-1 (HSV-1) latency-associated transcript (LAT) results in exhaustion of virus-specific CD8+ T cells in latently-infected trigeminal ganglia (TG). In this study we sought to determine if this impairment may involve LAT directly and/or indirectly interfering with DC maturation. We found that a small number of HSV-1 antigen-positive DCs are present in the TG of latently-infected CD11c/eYFP mice; however, this does not imply that these DCs are acutely or latently infected. Some CD8 + T cells are adjacent to DCs, suggesting possible interactions. It has previously been shown that wild-type HSV-1 interferes with DC maturation. Here we show for the first time that this is associated with LAT expression, since compared to LAT ( -) virus: (1) LAT ( + ) virus interfered with expression of MHC class I and the co-stimulatory molecules CD80 and CD86 on the surface of DCs; (2) LAT ( + ) virus impaired DC production of the proinflammatory cytokines IL-6, IL-12, and TNF-a; and (3) DCs infected in vitro with LAT ( + ) virus had significantly reduced the ability to stimulate HSV-specific CD8 + T cells. While a similar number of DCs was found in LAT ( + ) and LAT ( -) latently-infected TG of CD11c/eYFP transgenic mice, more HSV-1 Ag-positive DCs and more exhausted CD8 T cells were seen with LAT ( + ) virus. Consistent with these findings, HSV-specific cytotoxic CD8 + T cells in the TG of mice latently-infected with LAT ( + ) virus produced less IFN-c and TNF-a than those from TG of LAT ( -) infected mice. Together, these results suggest a novel immune-evasion mechanism whereby the HSV-1 LAT increases the number of HSV-1 Ag-positive DCs in latently-infected TG, and interferes with DC phenotypic and functional maturation. The effect of LAT on TG-resident DCs may contribute to the reduced function of HSVspecific CD8 + T cells in the TG of mice latently infected with LAT ( + ) virus.

CR1 (CD35, the C3b/C4b receptor) is a widely distributed membrane glycoprotein with a unique cluster conformation on the surface of erythrocytes (E). CR1 on E is responsible for the transport of immune complexes (IC) to liver and spleen. As a cofactor of the C3b cleavage by factor I, CR1 is also a potent inhibitor of C activation and inflammation. In some diseases (systemic lupus erythematosus, hemolytic anemia, AIDS, etc.) an acquired low level of CR1 on E has been observed, leading to an impaired clearance of IC. The aim of this study was to design a heterofunctional molecule that will bind to E and restore a normal or a supranormal CR1 density on E that could mimic the unique distribution pattern of CR1 on normal E. For that purpose a new multimerizing system based on the properties of the C-terminal part of the α-chain of the C4 binding protein (C4bp) was used. We first produced a multimeric soluble CR1 that proved to be a better inhibitor of in vitro C activation than the monomeric form of CR1, then a heteromultimeric molecule made of CR1 and single-chain Fv anti-Rh(D) valences able to attach E and providing E with as much as a 10-fold increase in CR1 density with the same CR1 distribution pattern as native E. CR1/single-chain Fv anti-Rh(D)-treated E were able in vitro to attach as many opsonized IC as native E. These data open the way for future use of multimeric and heteromultimeric forms of soluble recombinant CR1 as therapy of IC diseases.

Considering the limited success of the recent herpes clinical vaccine trial, new vaccine strategies are needed. Infections with HSV-1 and HSV-2 in the majority of men and women are usually asymptomatic and result in lifelong viral latency in neurons of sensory ganglia. However, in a minority of men and women, spontaneous HSV reactivation can cause recurrent disease (i.e., symptomatic individuals). Our recent findings show that T cells from symptomatic and asymptomatic men and women (i.e., those with and without recurrences, respectively) recognize different herpes epitopes. This finding breaks new ground and opens new doors to assess a new vaccine strategy: mucosal immunization with HSV-1 and HSV-2 epitopes that induce strong in vitro CD4 and CD8 T-cell responses from peripheral blood mononuclear cells derived from asymptomatic men and women (designated here as ‘asymptomatic’ protective epitopes) could boost local and systemic ‘natural’ protective immunity, induced by natural infection. Here we highlight the rationale and the future of our emerging asymptomatic T-cell epitope-based mucosal vaccine strategy to decrease recurrent herpetic disease.

A significantly larger repertoire of dysfunctional (exhausted) HSV-specific CD8(+)T cells were found in the TG of HLA transgenic rabbits latently infected with wild-type HSV-1 or with LAT-rescued mutant (i.e., LAT(+)TG) than in a more restricted repertoire of functional HSV-specific CD8(+)T cells in the TG of HLA transgenic rabbits latently infected with LAT-null mutant (i.e., LAT(-)TG). These findings suggest that the HSV-1 LAT locus interferes with the host cellular immune response by shaping a broader repertoire of exhausted HSV-specific CD8(+)T cells within the latency/reactivation TG site.

Current immunofluorescence protocols are limited as they do not provide reliable antibody staining within large tissue volumes (mm3) and cannot localise and quantify multiple antigens or cell populations in the same tissue at high resolution. To address this limitation, we have developed an approach to three-dimensionally visualise large tissue volumes (mm3) at high resolution (<1 µm) and with multiple antigen labelling, for volumetric and quantitative analysis. This is made possible through computer reconstruction of serial sectioned and sequentially immunostained butyl-methyl methacrylate (BMMA) embedded tissue. Using this novel immunofluorescent computed tomography (ICT) approach, we have three-dimensionally reconstructed part of the murine lower eyelid that contains the meibomian gland and localised cell nuclei (DAPI), Ki67 and cytokeratin 1 (CK1), as well as performing non-linear optical (NLO) microscopy imaging of collagen, to assess cell density, cell proliferation, gland keratinisation and gland volume respectively. Antigenicity was maintained after four iterative stains on the same tissue, suggesting that there is no defined limit to the number of antigens that can be immunostained for reconstruction, as long as the sections remain intact and the previous antibody has been successfully eluted. BMMA resin embedding also preserved fluorescence of transgenic proteins. We propose that ICT may provide valuable high resolution, three-dimensional biological maps of multiple biomolecules within a single tissue or organ to better characterise and quantify tissue structure and function.