Efficacy and Safety of Amenamevir, a Helicase-Primase Inhibitor for the Treatment of Acyclovir-Resistant Herpes Simplex Virus 1 Keratitis.

PURPOSE: The purpose of this study was to describe the efficacy and tolerance of amenamevir (AMNV), an inhibitor of the viral helicase-primase, for the treatment of recalcitrant herpes simplex keratitis (HSK) caused by acyclovir-resistant (ACVR) herpes simplex virus 1 strains. METHODS: In this retrospective case series, 6 consecutive patients with HSK caused by an ACVR herpes simplex virus 1 strain with a failure of conventional antiviral therapy were included after having been treated with AMNV (there was no control group of comparable patients for whom previous treatment would have been continued despite its inefficacy). Medical files were assessed for clinical data including reason(s) for AMNV introduction (frequent recurrences despite appropriate preventive antiviral treatment and/or clinical resistance to suppressive antiviral treatment of an ongoing clinical relapse), genotypical resistance to herpes simplex virus 1 documentation, immune status, clinical types and number of HSK episodes before and during AMNV treatment, adverse effects observed during AMNV treatment, and best corrected visual acuity. RESULTS: Of 6 patients, 4 (66%) did not experience a single recurrence during AMNV therapy while 2 others had recurrences (1 over 24 months of treatment and 2 over 23 months, ie two-fold less frequently than with conventional preventive treatment). On the overall history of these 6 patients, AMNV appeared to be associated with a reduction in HSK recurrences, with a mean of only 0.02 ± 0.04 episodes/month during follow-up under AMNV as compared to 0.14 ± 0.04 episodes/month in the year preceding AMNV introduction (P = 0.03). Improvement in vision acuity was also observed (mean best corrected visual acuity 0.17 ± 0.12 logarithm of the minimum angle of resolution at the end of follow-up vs. 0.30 ± 0.35 before AMNV onset), albeit nonsignificant probably due to the limited number of patients (P = 0.38). Neither clinical nor biological adverse effects were observed while under AMNV during the follow-up (16.5 ± 5.8 months). CONCLUSIONS: Although there was no control group, AMNV may be a valuable option to reduce ACVR HSK recurrences.

Identification of macaque dendritic cell precursors in blood and tissue reveals their dysregulation in early SIV infection.

Distinct dendritic cell (DC) subsets play important roles in shaping immune responses. Circulating DC precursors (pre-DCs) are more susceptible to HIV infection in vitro, which may explain the inefficiency of immune responses against HIV. However, the interplay between HIV and pre-DC is not defined in vivo. We identify human pre-DC equivalents in the cynomolgus macaque and then analyze their dynamics during simian immunodeficiency virus (SIV) infection to illustrate a sharp decrease of blood pre-DCs in early SIV infection and accumulation in lymph nodes (LNs), where they neglect to upregulate CD83/CD86 or MHC-II. Additionally, SIV infection attenuates the capacity of stimulated LN pre-DCs to produce IL-12p40. Analysis of HIV cohorts provides correlation between costimulatory molecule expression on pre-DCs and T cell activation in spontaneous HIV controllers. These findings pinpoint certain dynamics and functional changes of pre-DCs during SIV infection, providing a deeper understanding of immune dysregulation mechanisms elicited in people living with HIV.

Integrative temporal multi-omics reveals uncoupling of transcriptome and proteome during human T cell activation.

Engagement of the T cell receptor (TCR) triggers molecular reprogramming leading to the acquisition of specialized effector functions by CD4 helper and CD8 cytotoxic T cells. While transcription factors, chemokines, and cytokines are known drivers in this process, the temporal proteomic and transcriptomic changes that regulate different stages of human primary T cell activation remain to be elucidated. Here, we report an integrative temporal proteomic and transcriptomic analysis of primary human CD4 and CD8 T cells following ex vivo stimulation with anti-CD3/CD28 beads, which revealed major transcriptome-proteome uncoupling. The early activation phase in both CD4 and CD8 T cells was associated with transient downregulation of the mRNA transcripts and protein of the central glucose transport GLUT1. In the proliferation phase, CD4 and CD8 T cells became transcriptionally more divergent while their proteome became more similar. In addition to the kinetics of proteome-transcriptome correlation, this study unveils selective transcriptional and translational metabolic reprogramming governing CD4 and CD8 T cell responses to TCR stimulation. This temporal transcriptome/proteome map of human T cell activation provides a reference map exploitable for future discovery of biomarkers and candidates targeting T cell responses.

Ocular surface toxicities associated with modern anticancer therapies.

Cancer treatments have recently shifted from broad-spectrum cytotoxic therapies to more focused treatments, maximizing anticancerous activity while reducing toxicity to healthy cells. These modern anticancer therapies (MATs) encompass a wide range of innovative molecules that include immune checkpoint inhibitors and other targeted anticancer therapies, comprising antibody drug conjugates and inhibitors of signal transduction. Some MATs are associated with ocular surface adverse events that can cause severe discomfort and even lead to loss of vision. While these complications remain rare, they are probably underreported. It is likely that both oncologists and ophthalmologists will come across MATs-associated ocular surface-adverse events in their practices, owing to the increasing number of patients being treated with MATs. Rapid identification of ocular surface-adverse events is crucial, as early intervention can manage these conditions to avoid vision loss and reduce negative impacts on quality of life. We discuss characteristics of ocular surface pathologies attributed to MATs, describe the suspected underlying pathophysiological mechanisms, and outline the main lines of treatment.

Diagnostic performance of real-time quantitative PCR in tear samples in various subtypes of herpes simplex keratitis.

Diagnosis of herpes simplex keratitis (HSK) is mostly based on clinical findings, yet biological confirmation supports management of challenging cases. This study evaluated the place of real-time quantitative PCR (RT-qPCR) on tear samplings in the management of HSK. Clinical records of patients who underwent tear sampling tested by RT-qPCR for herpes simplex virus type 1 for an acute episode of corneal inflammation or defect between January 2013 and December 2021 were retrospectively reviewed, and results were compared to clinical diagnosis (i.e., HSK or not) based on biomicroscopic findings and medical history. Of 465 tested tear samples from 364 patients, a clinical diagnosis of active (ongoing) HSK was recorded in 240 cases, among which 76 were RT-qPCR positive (global sensitivity of 31.6%, specificity of 99.5%). Sensitivity of RT-qPCR was higher in epithelial (97.4%) and stromal keratitis with ulceration (48.7%), compared to other types of HSK (23.5% in keratouveitis, 13.6% in endotheliitis, 11.1% in postherpetic neurotrophic keratopathy, and 8.1% in stromal keratitis without ulceration). The highest viral loads were detected from epithelial and stromal keratitis with ulceration, while in HSK with no epithelial involvement, the viral load detected was 196-fold lower, on average. The proportion of clinically characterized HSK patients with negative tear samples was higher in patients receiving antiviral treatment (P < 0.0001). RT-qPCR, performed on tear samples, can help in confirming diagnosis in case of presumed HSK, including clinical forms with no obvious epithelial involvement. The sensitivity of tear sampling is much higher whenever epithelial keratitis is present.

Herpes simplex virus, varicella-zoster virus and cytomegalovirus keratitis: Facts for the clinician.

Keratitis due to Herpes simplex virus (HSK), Varicella-Zoster virus (VZK) and Cytomegalovirus remains a frequent source of concern for many ophthalmologists. They are a frequent cause of emergency consultations at eye care centers and carry the risk of permanent loss of visual acuity or visual quality and/or chronic neurotrophic keratitis, resulting in a significant decrease in the quality of life. HSK and VZK can affect the corneal epithelium, stroma, or endothelium or a combination of layers. In contrast, most cases of CMV keratitis present as isolated endothelitis (CMVE), a clinical entity that has been described within the last 2 decades. These three types of viral keratitis are characterized by a high frequency of recurrences and each new episode increases the risk of sequelae. Hence, ophthalmologists must adapt the treatment to the clinical presentation of each recurrent episode in order to mitigate the immediate consequences of viral replication and the immune response on corneal transparency. In patients with frequent recurrences, preventive long-term antiviral treatment is strongly recommended. However, in some rare cases, continuous exposure to antivirals may promote the emergence of resistant viral strains, which can be difficult to manage. In the future, the introduction of new antiviral drugs, with differing modes of action compared to current medical therapy, could be an alternative until a truly effective preventive solution, such as a vaccine, is available.

Associations between ocular and extra-ocular assessment in primary Sjögren's syndrome.

OBJECTIVES: To assess associations between ophthalmological features and the main systemic biomarkers of primary Sjögren's Syndrome (pSS), and to identify systemic biomarkers associated with severe keratoconjunctivitis sicca (KCS) in pSS patients. METHODS: In this cross-sectional study, data was retrospectively extracted from the monocentric cohort of the French reference centre for pSS. We analysed data from the initial visit of patients admitted for suspicion of pSS and included patients validating pSS ACR/EULAR classification criteria. Ophthalmological assessment included Schirmer's test, tear break-up time, ocular staining score (OSS), and visual analogue scale (DED-VAS) for dry eye disease (DED) symptoms. Results of minor salivary gland biopsy, unstimulated whole salivary flow rate, anti-SSA/Ro antibodies, anti-SSB/La antibodies, and rheumatoid factor (RF) were collected. RESULTS: A total of 253 patients (245 females) with confirmed pSS, aged 56.6±13.0 years, were included, among which 37% had severe KCS. Multivariate analysis showed that the presence of anti-SSA/Ro antibodies, anti-SSB/La antibodies and RF were associated with conjunctival OSS (odds ratio-OR-=1.25 per OSS unit increase; confidence interval-CI-95%=1.05-1.49; P=0.01; OR=1.31 per OSS unit increase; CI95%=1.09-1.58, P=0.002, and OR=1.34 per OSS unit increase; CI95%=1.12-1.59; P=0.001, respectively). Both anti-SSB/La antibodies and DED-VAS ≥ 5 were significantly associated with severe KCS (OR=2.03; CI95%=1.03-4.00; P<0.05 and OR=2.52, CI95%=1.31-4.90; P<0.01, respectively). CONCLUSION: Association between conjunctival OSS and systemic biomarkers of pSS indicate the crucial importance of conjunctival staining when pSS is suspected as a cause of DED. Conversely, patients with anti-SSB and DED-VAS ≥ 5 features should be prioritized for extensive evaluation by an ophthalmologist due to their association with severe KCS.

Initial TK-deficient HSV-1 infection in the lip alters contralateral lip challenge immune dynamics.

Primary infection with herpes simplex type 1 (HSV-1) occurring around the mouth and nose switches rapidly to lifelong latent infection in sensitive trigeminal ganglia (TG) neurons. Sporadic reactivation of these latent reservoirs later in life is the cause of acute infections of the corneal epithelium, which can cause potentially blinding herpes simplex keratitis (HSK). There is no effective vaccine to protect against HSK, and antiviral drugs provide only partial protection against recurrences. We previously engendered an acute disease-free, non-reactivating latent state in mice when challenged with virulent HSV-1 in orofacial mucosa, by priming with non-neurovirulent HSV-1 (TKdel) before the challenge. Herein, we define the local immune infiltration and inflammatory chemokine production changes after virulent HSV-1 challenge, which were elicited by TKdel prime. Heightened immunosurveillance before virulent challenge, and early enhanced lymphocyte-enriched infiltration of the challenged lip were induced, which corresponded to attenuation of inflammation in the TG and enhanced viral control. Furthermore, classical latent-phase T cell persistence around latent HSV-1 reservoirs were severely reduced. These findings identify the immune processes that are likely to be responsible for establishing non-reactivating latent HSV-1 reservoirs. Stopping reactivation is essential for development of efficient vaccine strategies against HSV-1.

Rubella virus-associated uveitis: The essentiality of aqueous humor virological analysis.

AIMS / BACKGROUND: Rubella virus-associated uveitis (RVAU) classically presents with the clinical features of Fuchs uveitis syndrome (FUS). We report a series RVAU, and discuss the relevance of available diagnostic strategies, and how vaccination could potentially prevent disease. METHODS: We retrospectively included patients with RV-positive aqueous humor (AH) with RT-PCR and/or intraocular RV-IgG production, between January 2014 and December 2019. RV-IgG titers from AH and serum were compared with other virus-specific IgG titers (VZV and/or CMV and/or HSV-1), to determine the derived Goldmann-Witmer coefficient (GWC'). Clinical findings at presentation and during follow-up are reported, as well as the anti-RV vaccination status. RESULTS: All 13 included patients demonstrated intraocular synthesis of RV-IgG (median GWC': 9.5; 3.2-100). RV-RNA was detected in one patient while PCR results were negative for other HSV1, VZV and CMV. The mean delay in diagnosis was 13 ± 12.6 years, with an initial presentation of FUS in only 3 patients (23%). Only four patients had been vaccinated, but all after the recommended age. CONCLUSION: As RVAU is a pleiomorphic entity, virological analysis (RV RT-PCR and GWC') of aqueous humor is essential to improve the diagnosis and management of this entity. Improper vaccination against RV appears to be implicated in RVAU.

DROSHA but not DICER is required for human haematopoietic stem cell function.

OBJECTIVES: DROSHA and DICER have central roles in the biogenesis of microRNAs (miRNAs). However, we previously showed that in the murine system, DROSHA has an alternate function where it directly recognises and cleaves protein-coding messenger (m)RNAs and this is critical for safeguarding the pluripotency of haematopoietic stem cells (HSCs). Maintenance of murine HSC function is dependent on DROSHA-mediated cleavage of two mRNAs, Myl9 and Todr1. The goal of this study is to determine whether this pathway is conserved in human HSCs. METHODS: DROSHA and DICER were knocked down in human cord blood CD34+ HSCs with short hairpin RNAs. The function of HSCs was analysed in vitro and in humanised mice. Analysis of mRNA cleavage was performed by capture of 5' phosphorylated RNAs. RESULTS: Consistent with murine HSCs, DROSHA knockdown impaired the differentiation of human HSCs in vitro and engraftment into humanised mice, whereas DICER knockdown had no impact. DROSHA cleaves the MYL9 mRNA in human HSCs and DROSHA deficiency resulted in the accumulation of the mRNA. However, ectopic expression of MYL9 did not impair human HSC function. We were unable to identify a human homolog of Todr1. CONCLUSION: A miRNA-independent function of DROSHA is critical for the function of human HSCs. DROSHA directly recognises and degrades mRNAs in humans HSCs. However, unlike in murine HSCs, the degradation of the MYL9 mRNA alone is not critical for human HSC function. Therefore, DROSHA must be inhibiting other targets and/or has another miRNA-independent function that is essential for safeguarding the pluripotency of human HSCs.

Acyclovir-Resistant Herpes Simplex Virus 1 Keratitis: A Concerning and Emerging Clinical Challenge.

PURPOSE: To describe the clinical and virological profiles of patients with herpes simplex keratitis (HSK) caused by acyclovir-resistant (ACVR) strains of herpes simplex virus 1 (HSV-1). DESIGN: Multicenter retrospective case series. METHODS: HSV-1 resistance to ACV was confirmed using sequencing of genes encoding HSV-1 thymidine kinase (TK) and DNA polymerase (DNA pol). Data were collected on the number of HSK episodes before and after the diagnosis of resistance, ocular findings including the type of HSK, immune status of patients, antiviral treatments, and HSV-1 genotypic resistance profiles. RESULTS: This study evaluated 18 HSK patients (13 male and 5 female, aged 66.8 ± 4.7 years) with ACVR HSV-1-positive ocular samples. Genotypic resistance testing was performed because of frequent recurrences despite adequate antiviral prophylaxis (AVP) (n = 13, 72%), or poor response to suppressive antiviral therapy (n = 5, 28%). Resistance mutations were found in the TK (n = 15, 83%) or in the DNA pol gene (n = 3, 17%). Prior to the diagnosis of resistance, the duration of disease was 29.8 ± 20.4 years, with more than 10 HSK recurrences in 15 patients (83%). The number of recurrences between the first episode and the diagnosis of resistance was significantly lower in immunocompromised patients (n = 6, 33%) than in immunocompetent patients (n = 12; 67%) (11.5 ± 4.9 vs 16.4 ± 1.9, P = .05). CONCLUSION: HSV-1 resistance to ACV must be suspected in HSK patients with recurrences despite AVP and/or in cases that respond poorly to a suppressive antiviral regimen. Immunocompromised patients and/or those with longstanding disease may be particularly at risk for developing resistance.

Synthetic hookworm-derived peptides are potent modulators of primary human immune cell function that protect against experimental colitis in vivo.

The prevalence of autoimmune diseases is on the rise globally. Currently, autoimmunity presents in over 100 different forms and affects around 9% of the world's population. Current treatments available for autoimmune diseases are inadequate, expensive, and tend to focus on symptom management rather than cure. Clinical trials have shown that live helminthic therapy can decrease chronic inflammation associated with inflammatory bowel disease and other gastrointestinal autoimmune inflammatory conditions. As an alternative and better controlled approach to live infection, we have identified and characterized two peptides, Acan1 and Nak1, from the excretory/secretory component of parasitic hookworms for their therapeutic activity on experimental colitis. We synthesized Acan1 and Nak1 peptides from the Ancylostoma caninum and Necator americanus hookworms and assessed their structures and protective properties in human cell-based assays and in a mouse model of acute colitis. Acan1 and Nak1 displayed anticolitic properties via significantly reducing weight loss and colon atrophy, edema, ulceration, and necrosis in 2,4,6-trinitrobenzene sulfonic acid-exposed mice. These hookworm peptides prevented mucosal loss of goblet cells and preserved intestinal architecture. Acan1 upregulated genes responsible for the repair and restitution of ulcerated epithelium, whereas Nak1 downregulated genes responsible for epithelial cell migration and apoptotic cell signaling within the colon. These peptides were nontoxic and displayed key immunomodulatory functions in human peripheral blood mononuclear cells by suppressing CD4+ T cell proliferation and inhibiting IL-2 and TNF production. We conclude that Acan1 and Nak1 warrant further development as therapeutics for the treatment of autoimmunity, particularly gastrointestinal inflammatory conditions.

Genetic Bias, Diversity Indices, Physiochemical Properties and CDR3 Motifs Divide Auto-Reactive from Allo-Reactive T-Cell Repertoires.

The distinct properties of allo-reactive T-cell repertoires are not well understood. To investigate whether auto-reactive and allo-reactive T-cell repertoires encoded distinct properties, we used dextramer enumeration, enrichment, single-cell T-cell receptor (TCR) sequencing and multiparameter analysis. We found auto-reactive and allo-reactive T-cells differed in mean ex vivo frequency which was antigen dependent. Allo-reactive T-cells showed clear differences in TCR architecture, with enriched usage of specific T-cell receptor variable (TRBJ) genes and broader use of T-cell receptor variable joining (TRBJ) genes. Auto-reactive T-cell repertoires exhibited complementary determining regions three (CDR3) lengths using a Gaussian distribution whereas allo-reactive T-cell repertoires exhibited distorted patterns in CDR3 length. CDR3 loops from allo-reactive T-cells showed distinct physical-chemical properties, tending to encode loops that were more acidic in charge. Allo-reactive T-cell repertoires differed in diversity metrics, tending to show increased overall diversity and increased homogeneity between repertoires. Motif analysis of CDR3 loops showed allo-reactive T-cell repertoires differed in motif preference which included broader motif use. Collectively, these data conclude that allo-reactive T-cell repertoires are indeed different to auto-reactive repertoires and provide tangible metrics for further investigations and validation. Given that the antigens studied here are overexpressed on multiple cancers and that allo-reactive TCRs often show increased ligand affinity, this new TCR bank also has translational potential for adoptive cell therapy, soluble TCR-based therapy and rational TCR design.

Peripheral ulcerative keratitis in rheumatoid arthritis patients taking tocilizumab: paradoxical manifestation or insufficient efficacy?

OBJECTIVES: Peripheral ulcerative keratitis (PUK) is a severe corneal condition associated with uncontrolled RA. Tocilizumab (TCZ) is used to control RA, however, episodes of paradoxical ocular inflammation have been reported in TCZ-treated patients. We report a case series of PUK in TCZ-treated RA patients with ophthalmological and systemic findings and discuss the potential underlying mechanisms. METHODS: Four patients (six eyes), 47-62 years of age, were included. At the onset of PUK, the median duration of RA was 13 years [interquartile range (IQR) 3-13] and the median treatment with TCZ was 9 months (IQR 3-14). Two patients had active disease [28-joint DAS (DAS28) >3.2] and the disease was controlled in two patients (DAS28 ≤3.2). RESULTS: TCZ was initially replaced by another immunomodulatory treatment in all patients and later reintroduced in two patients without PUK recurrence. Corneal inflammation was controlled in all cases with local and systemic treatments, with severe visual loss in one eye. CONCLUSION: PUK may occur in patients with long-standing RA after a switch to TCZ and can be interpreted, depending on the context, as insufficient efficacy or a paradoxical manifestation. These cases highlight the urgent need for reliable biomarkers of the efficacy and paradoxical reactions of biologics.

Using pre-existing social networks to determine the burden of disease and real-life needs in rare diseases: the example of Thygeson's superficial punctate keratitis.

BACKGROUND: Thygeson's superficial punctate keratitis (TSPK) is a rare and still poorly understood disease of the ocular surface, responsible for recurrent episodes of photophobia and eye pain. While TSPK is considered as a benign condition, a subset of patients has frequent recurrences or even chronic disease, two situations in which there are currently no therapeutic guidelines. We used a preexisting Facebook TSPK patient support group to assess the clinical journey and the burden of disease of TSPK. RESULTS: An online survey was sent to the patient support group. The first part of the questionnaire gathered information on demographics and the patient's clinical journey [diagnostic modalities, symptoms, duration and frequency of recurrent episodes (RE), efficacy and tolerance to treatments]. The second part focused on quality of life (QoL) using the Ocular Surface Disease-QoL (OSD-QoL) questionnaire. Seventy-two patients out of 595 members of the support group completed the questionnaire during the 3-months study period. Eighty percent of patients developed symptoms before 30 years old, and 47% reported a delay in the diagnosis above 1 year. Sixty percent of patients reported over 5 RE yearly, and 18% of RE lasted more than 3 months. Forty percent of all patients used cyclosporine eyedrops (50% of those with > 5 episodes/year) and it was perceived as effective by 72% of these patients. The impact on daily life activities was judged as severe by 22% of patients, while 38% reported reduced professional activity and 80% were deeply saddened by their eye condition. CONCLUSION: TSPK patients may present with frequent recurrences and/or chronic disease, that result in a severe impact on QoL, and an off-label use of topical immunomodulatory eye drops, suggesting the urgent need for controlled studies. The utility of using social networks for rare ophthalmic disease research includes, faster data collection, data from patients across the globe, and also raises relevant questions about their real needs.

Ocular Manifestations of West Nile Virus.

Ocular manifestations are a feature of West Nile virus infection. They mostly occur in association with severe neuroinvasive disease. Linear chorioretinitis is suggestive of the diagnosis and may raise diagnostic suspicion when associated with evocative systemic signs, and in an epidemic context. Various other less specific inflammatory ocular manifestations have been reported, including anterior uveitis, occlusive retinal vasculitis, optic neuritis, and diplopia. The pathophysiology of ocular disease remains unclear, but it reflects the neuroinvasiveness of the disease. Although ocular involvement most often resolves without visual sequelae, some patients may have permanent loss of vision, adding to the need for the development of a specific treatment and/or vaccines.

Human CLEC9A antibodies deliver NY-ESO-1 antigen to CD141+ dendritic cells to activate naïve and memory NY-ESO-1-specific CD8+ T cells.

BACKGROUND: Dendritic cells (DCs) are crucial for the efficacy of cancer vaccines, but current vaccines do not harness the key cDC1 subtype required for effective CD8+ T-cell-mediated tumor immune responses. Vaccine immunogenicity could be enhanced by specific delivery of immunogenic tumor antigens to CD141+ DCs, the human cDC1 equivalent. CD141+ DCs exclusively express the C-type-lectin-like receptor CLEC9A, which is important for the regulation of CD8+ T cell responses. This study developed a new vaccine that harnesses a human anti-CLEC9A antibody to specifically deliver the immunogenic tumor antigen, NY-ESO-1 (New York esophageal squamous cell carcinoma 1), to human CD141+ DCs. The ability of the CLEC9A-NY-ESO-1 antibody to activate NY-ESO-1-specific naïve and memory CD8+ T cells was examined and compared with a vaccine comprised of a human DEC-205-NY-ESO-1 antibody that targets all human DCs. METHODS: Human anti-CLEC9A, anti-DEC-205 and isotype control IgG4 antibodies were genetically fused to NY-ESO-1 polypeptide. Cross-presentation to NY-ESO-1-epitope-specific CD8+ T cells and reactivity of T cell responses in patients with melanoma were assessed by interferon γ (IFNγ) production following incubation of CD141+ DCs and patient peripheral blood mononuclear cells with targeting antibodies. Humanized mice containing human DC subsets and a repertoire of naïve NY-ESO-1-specific CD8+ T cells were used to investigate naïve T cell priming. T cell effector function was measured by expression of IFNγ, MIP-1ß, tumor necrosis factor and CD107a and by lysis of target tumor cells. RESULTS: CLEC9A-NY-ESO-1 antibodies (Abs) were effective at mediating delivery and cross-presentation of multiple NY-ESO-1 epitopes by CD141+ DCs for activation of NY-ESO-1-specific CD8+ T cells. When benchmarked to NY-ESO-1 conjugated to an untargeted control antibody or to anti-human DEC-205, CLEC9A-NY-ESO-1 was superior at ex vivo reactivation of NY-ESO-1-specific T cell responses in patients with melanoma. Moreover, CLEC9A-NY-ESO-1 induced priming of naïve NY-ESO-1-specific CD8+ T cells with polyclonal effector function and potent tumor killing capacity in vitro. CONCLUSIONS: These data advocate human CLEC9A-NY-ESO-1 Ab as an attractive strategy for specific targeting of CD141+ DCs to enhance tumor immunogenicity in NY-ESO-1-expressing malignancies.

Human CLEC9A antibodies deliver Wilms' tumor 1 (WT1) antigen to CD141+ dendritic cells to activate naïve and memory WT1-specific CD8+ T cells.

OBJECTIVES: Vaccines that prime Wilms' tumor 1 (WT1)-specific CD8+ T cells are attractive cancer immunotherapies. However, immunogenicity and clinical response rates may be enhanced by delivering WT1 to CD141+ dendritic cells (DCs). The C-type lectin-like receptor CLEC9A is expressed exclusively by CD141+ DCs and regulates CD8+ T-cell responses. We developed a new vaccine comprising a human anti-CLEC9A antibody fused to WT1 and investigated its capacity to target human CD141+ DCs and activate naïve and memory WT1-specific CD8+ T cells. METHODS: WT1 was genetically fused to antibodies specific for human CLEC9A, DEC-205 or ß-galactosidase (untargeted control). Activation of WT1-specific CD8+ T-cell lines following cross-presentation by CD141+ DCs was quantified by IFNγ ELISPOT. Humanised mice reconstituted with human immune cell subsets, including a repertoire of naïve WT1-specific CD8+ T cells, were used to investigate naïve WT1-specific CD8+ T-cell priming. RESULTS: The CLEC9A-WT1 vaccine promoted cross-presentation of WT1 epitopes to CD8+ T cells and mediated priming of naïve CD8+ T cells more effectively than the DEC-205-WT1 and untargeted control-WT1 vaccines. CONCLUSIONS: Delivery of WT1 to CD141+ DCs via CLEC9A stimulates CD8+ T cells more potently than either untargeted delivery or widespread delivery to all Ag-presenting cells via DEC-205, suggesting that cross-presentation by CD141+ DCs is sufficient for effective CD8+ T-cell priming in humans. The CLEC9A-WT1 vaccine is a promising candidate immunotherapy for malignancies that express WT1.

Herpes Simplex Virus 1 Replication, Ocular Disease, and Reactivations from Latency Are Restricted Unilaterally after Inoculation of Virus into the Lip.

Ocular herpes simplex keratitis (HSK) is a consequence of viral reactivations from trigeminal ganglia (TG) and occurs almost exclusively in the same eye in humans. In our murine oro-ocular (OO) model, herpes simplex virus 1 (HSV-1) inoculation in one side of the lip propagates virus to infect the ipsilateral TG. Replication here allows infection of the brainstem and infection of the contralateral TG. Interestingly, HSK was observed in our OO model only from the eye ipsilateral to the site of lip infection. Thus, unilateral restriction of HSV-1 may be due to differential kinetics of virus arrival in the ipsilateral versus contralateral TG. We inoculated mice with HSV-1 reporter viruses and then superinfected them to monitor changes in acute- and latent-phase gene expression in TG after superinfection compared to the control (single inoculation). Delaying superinfection by 4 days after initial right lip inoculation elicited failed superinfecting-virus gene expression and eliminated clinical signs of disease. Initial inoculation with thymidine kinase-deficient HSV-1 (TKdel) completely abolished reactivation of wild-type (WT) superinfecting virus from TG during the latent stage. In light of these seemingly failed infections, viral genome was detected in both TG. Our data demonstrate that inoculation of HSV-1 in the lip propagates virus to both TG, but with delay in reaching the TG contralateral to the side of lip infection. This delay is responsible for restricting viral replication to the ipsilateral TG, which abrogates ocular disease and viral reactivations from the contralateral side. These observations may help to understand why HSK is observed unilaterally in humans, and they provide insight into vaccine strategies to protect against HSK.IMPORTANCE Herpetic keratitis (HK) is the leading cause of blindness by an infectious agent in the developed world. This disease can occur after reactivation of herpes simplex virus 1 in the trigeminal ganglia, leading to dissemination of virus to, and infection of, the cornea. A clinical paradox is evidenced by the bilateral presence of latent viral genomes in both trigeminal ganglia, while for any given patient the disease is unilateral with recurrences in a single eye. Our study links the kinetics of early infection to unilateral disease phenomenon and demonstrates protection against viral reactivation when kinetics are exploited. Our results have direct implications in the understanding of human disease pathogenesis and immunotherapeutic strategies for the treatment of HK and viral reactivations.