Development of AAV-delivered broadly neutralizing anti-human ACE2 antibodies against SARS-CoV-2 variants.

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in the emergence of new variants that are resistant to existing vaccines and therapeutic antibodies, has raised the need for novel strategies to combat the persistent global COVID-19 epidemic. In this study, a monoclonal anti-human angiotensin-converting enzyme 2 (hACE2) antibody, ch2H2, was isolated and humanized to block the viral receptor-binding domain (RBD) binding to hACE2, the major entry receptor of SARS-CoV-2. This antibody targets the RBD-binding site on the N terminus of hACE2 and has a high binding affinity to outcompete the RBD. In vitro, ch2H2 antibody showed potent inhibitory activity against multiple SARS-CoV-2 variants, including the most antigenically drifted and immune-evading variant Omicron. In vivo, adeno-associated virus (AAV)-mediated delivery enabled a sustained expression of monoclonal antibody (mAb) ch2H2, generating a high concentration of antibodies in mice. A single administration of AAV-delivered mAb ch2H2 significantly reduced viral RNA load and infectious virions and mitigated pulmonary pathological changes in mice challenged with SARS-CoV-2 Omicron BA.5 subvariant. Collectively, the results suggest that AAV-delivered hACE2-blocking antibody provides a promising approach for developing broad-spectrum antivirals against SARS-CoV-2 and potentially other hACE2-dependent pathogens that may emerge in the future.

Enrichment of Prevotella intermedia in human colorectal cancer and its additive effects with Fusobacterium nucleatum on the malignant transformation of colorectal adenomas.

BACKGROUND: Owing to the heterogeneity of microbiota among individuals and populations, only Fusobacterium nucleatum and Bacteroides fragilis have been reported to be enriched in colorectal cancer (CRC) in multiple studies. Thus, the discovery of additional bacteria contributing to CRC development in various populations can be expected. We aimed to identify bacteria associated with the progression of colorectal adenoma to carcinoma and determine the contribution of these bacteria to malignant transformation in patients of Han Chinese origin. METHODS: Microbiota composition was determined through 16S rRNA V3-V4 amplicon sequencing of autologous adenocarcinomas, adenomatous polyps, and non-neoplastic colon tissue samples (referred to as "tri-part samples") in patients with CRC. Enriched taxa in adenocarcinoma tissues were identified through pairwise comparison. The abundance of candidate bacteria was quantified through genomic quantitative polymerase chain reaction (qPCR) in tissue samples from 116 patients. Associations of candidate bacteria with clinicopathological features and genomic and genetic alterations were evaluated through odds ratio tests. Additionally, the effects of candidate bacteria on CRC cell proliferation, migration, and invasion were evaluated through the co-culture of CRC cells with bacterial cells or with conditioned media from bacteria. RESULTS: Prevotella intermedia was overrepresented in adenocarcinomas compared with paired adenomatous polyps. Furthermore, co-abundance of P. intermedia and F. nucleatum was observed in tumor tissues. More notably, the coexistence of these two bacteria in adenocarcinomas was associated with lymph node involvement and distant metastasis. These two bacteria also exerted additive effects on the enhancement of the migration and invasion abilities of CRC cells. Finally, conditioned media from P. intermedia promoted the migration and invasion of CRC cells. CONCLUSION: This report is the first to demonstrate that P. intermedia is enriched in colorectal adenocarcinoma tissues and enhances the migration and invasion abilities of CRC cells. Moreover, P. intermedia and F. nucleatum exert additive effects on the malignant transformation of colorectal adenomas into carcinomas. These findings can be used to identify patients at a high risk of malignant transformation of colorectal adenomas or metastasis of CRC, and they can accordingly be provided optimal clinical management.

Galectin-7 downregulation in lesional keratinocytes contributes to enhanced IL-17A signaling and skin pathology in psoriasis.

Psoriasis is a chronic inflammatory skin disease characterized by inflammatory cell infiltration, as well as hyperproliferation of keratinocytes in skin lesions, and is considered a metabolic syndrome. We found that the expression of galectin-7 is reduced in skin lesions of patients with psoriasis. IL-17A and TNF-α, 2 cytokines intimately involved in the development of psoriatic lesions, suppressed galectin-7 expression in human primary keratinocytes (HEKn cells) and the immortalized human keratinocyte cell line HaCaT. A galectin-7 knockdown in these cells elevated the production of IL-6 and IL-8 and enhanced ERK signaling when the cells were stimulated with IL-17A. Galectin-7 attenuated IL-17A-induced production of inflammatory mediators by keratinocytes via the microRNA-146a/ERK pathway. Moreover, galectin-7-deficient mice showed enhanced epidermal hyperplasia and skin inflammation in response to intradermal IL-23 injection. We identified fluvastatin as an inducer of galectin-7 expression by connectivity map analysis, confirmed this effect in keratinocytes, and demonstrated that fluvastatin attenuated IL-6 and IL-8 production induced by IL-17A. Thus, we validate a role of galectin-7 in the pathogenesis of psoriasis, in both epidermal hyperplasia and keratinocyte-mediated inflammatory responses, and formulate a rationale for the use of statins in the treatment of psoriasis.

Galectin-7 Regulates Keratinocyte Proliferation and Differentiation through JNK-miR-203-p63 Signaling.

Galectin-7, a member of the ß-galactoside-binding protein family, is primarily expressed in stratified epithelial cells, including keratinocytes. There is information in the literature suggesting a role for this protein in regulation of keratinocyte survival and growth, but the underlying mechanism remains relatively unknown. Moreover, its expression pattern in the epidermis suggests that it is also involved in the regulation of keratinocyte differentiation. Here, we demonstrate that galectin-7 knockdown results in reduced differentiation and increased proliferation of keratinocytes. Using microarray and deep-sequencing analyses, we found that galectin-7 positively and negatively regulates microRNA (miR)-203 and miR-146a expression, respectively. We show that galectin-7 regulates keratinocyte differentiation and proliferation through miR-203 but not miR-146a. A knockdown of either galectin-7 or miR-203 in keratinocytes increases expression of p63, an essential transcription factor involved in skin development. Rescue of miR-203 expression in a galectin-7 knockdown model reduces p63 expression to baseline. Increased galectin-7 expression upregulates c-Jun N-terminal kinase (JNK) protein levels, which is required for miR-203 expression. Finally, we establish that galectin-7 can be associated with JNK1 and protect it from ubiquitination and degradation. Thus, our data suggest an intracellular function of galectin-7: regulation of keratinocyte proliferation and differentiation through the JNK1-miR-203-p63 pathway.

Galectin-3 promotes HIV-1 budding via association with Alix and Gag p6.

Galectin-3 has been reported to regulate the functions of a number of immune cell types. We previously reported that galectin-3 is translocated to immunological synapses in T cells upon T-cell receptor engagement, where it associates with ALG-2-interacting protein X (Alix). Alix is known to coordinate with the endosomal sorting complex required for transport (ESCRT) to promote human immunodeficiency virus (HIV)-1 virion release. We hypothesized that galectin-3 plays a role in HIV-1 viral budding. Cotransfection of cells of the Jurkat T line with galectin-3 and HIV-1 plasmids resulted in increased HIV-1 budding, and suppression of galectin-3 expression by RNAi in Hut78 and primary CD4+ T cells led to reduced HIV-1 budding. We used immunofluorescence microscopy to observe the partial colocalization of galectin-3, Alix and Gag in HIV-1-infected cells. Results from co-immunoprecipitation experiments indicate that galectin-3 expression promotes Alix-Gag p6 association, whereas the results of Alix knockdown suggest that galectin-3 promotes HIV-1 budding through Alix. HIV-1 particles released from galectin-3-expressing cells acquire the galectin-3 protein in an Alix-dependent manner, with proteins primarily residing inside the virions. We also found that the galectin-3 N-terminal domain interacts with the proline-rich region of Alix. Collectively, these results suggest that endogenous galectin-3 facilitates HIV-1 budding by promoting the Alix-Gag p6 association.

Cancer immunotherapy using a membrane-bound interleukin-12 with B7-1 transmembrane and cytoplasmic domains.

Interleukin-12 (IL-12) has potent antitumor activity, but its clinical application is limited by severe systemic toxicity, which might be alleviated by the use of membrane-anchored IL-12. In the present study, a new membrane-bound IL-12 containing murine single-chain IL-12 and B7-1 transmembrane and cytoplasmic domains (scIL-12-B7TM) was constructed and its efficacy in cancer treatment examined and its protective antitumor mechanism investigated. Surface expression of scIL-12-B7TM on colon adenocarcinoma cells significantly inhibited the growth of subcutaneous tumors, suppressed lung metastasis, and resulted in local and systemic suppression of unmodified tumors. Intratumoral injection of an adenoviral vector encoding scIL-12-B7TM not only resulted in complete regression of a majority of local tumors, but also significantly suppressed the growth of distant, untreated tumors. Moreover, mice that had been treated with scIL-12-B7TM developed memory responses against subsequent tumor challenge. Immunohistochemical staining and in vivo depletion of lymphocyte subpopulations demonstrated that both CD8(+) T cells and CD4(+) T cells contributed to the antitumor activity of scIL-12-B7TM. Importantly, the potent antitumor activities of scIL-12-B7TM were achieved with only negligible amounts of IL-12 in the circulation. Our data demonstrate that cancer immunotherapy using membrane-bound IL-12 has the advantage of minimizing systemic IL-12 levels without compromising its antitumor efficacy.

Differential antitumor effect of interleukin-12 family cytokines on orthotopic hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most difficult cancers to treat. The interleukin (IL)-12 family cytokines, including IL-12, IL-23 and IL-27, display overlapping, but not redundant, roles in regulating lymphocyte subpopulations. IL-12 is known as a potent antitumor cytokine, whereas the results of the antitumor effect of IL-23 and IL-27 are inconsistent. The present study aimed to directly compare the relative antitumor efficacy of these three IL-12 family cytokines on HCC. METHODS: A murine orthotopic BNL HCC model, in which the tumor is located in an environment heavily populated with different lymphocyte subsets, was established. The hepatotropic adeno-associated virus serotype 8 (AAV8) vector was used to deliver the cytokine genes aiming to achieve sustained cytokine expression in the liver. RESULTS: AAV8/IL-12 treatment significantly reduced hepatic metastases and prolonged survival time, whereas treatment with AAV8/IL-23 or AAV8/IL-27 had only moderate antitumor effects at a high dose. The antitumor efficacy of these cytokines was positively correlated with their ability to regulate hepatic T cells, natural killer cells and natural killer T cells, with IL-12 greatly increasing the number and activation status of these cells, whereas IL-27 had no effect and IL-23 had a negative effect. AAV8/IL-12 treatment also resulted in a marked decrease in tumor vessel density, which was not observed with AAV8/IL-23 and AAV8/IL-27 treatment. CONCLUSIONS: The data obtained in the present study highlight the importance of local lymphocytes and anti-angiogenesis for influencing the antitumor activity of these three IL-12 family cytokines and suggest that IL-12 is the best candidate for treating HCC.

Treatment of hepatocellular carcinoma with adeno-associated virus encoding interleukin-15 superagonist.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, but effective therapies are still needed. The liver has been identified as an important immune organ and is heavily populated with various lymphocyte subsets known to play important roles in cancer immunosurveillance. We hypothesized that activation of hepatic lymphocytes by interleukin (IL)-15, a cytokine known for its ability to trigger proliferation and activation of natural killer (NK) cells, natural killer T cells, and memory CD8(+) T cells, might offer an alternative therapy for HCC. We employed hepatotropic adeno-associated virus serotype 8 (AAV8) to deliver an IL-15 superagonist (IL-15-IL-15RalphaS), consisting of IL-15 covalently linked to the N-terminal sushi domain of the IL-15 receptor alpha chain, to achieve local sustained cytokine expression in the liver environment. We observed that a single injection of AAV8 expressing IL-15-IL-15RalphaS, but not IL-15 alone, greatly expanded the number of hepatic mononuclear cells, mainly NK cells, for at least 21 days. AAV8/IL-15-IL-15RalphaS treatment generated potent antitumor activity in a liver metastatic murine HCC model (BNL cells), and significantly prolonged the survival time of treated animals. The antitumor effect depended mainly on NK cells, not on CD8(+) and CD4(+) T cells, because AAV8/IL-15-IL-15RalphaS treatment greatly enhanced the cytolytic activity of hepatic NK cells and depletion of NK cells abrogated the therapeutic effect. Importantly, no apparent liver toxicity was observed during AAV8/IL-15-IL-15RalphaS treatment. Together, our data demonstrate that AAV8-delivered IL-15-IL-15RalphaS provides an effective and safe therapy against metastatic HCC.

Electroporation-mediated IL-12 gene therapy in a transplantable canine cancer model.

Interleukin-12 (IL-12) is effective in treating many types of rodent tumors, but has been unsuccessful in most human clinical trials, suggesting that animal models of more clinical relevance are required for evaluating human cancer immunotherapy. Herein, we report on the effectiveness of gene therapy with plasmid encoding human IL-12 (pIL-12) through in vivo electroporation in the treatment of beagles with a canine tumor, the canine transmissible venereal tumor (CTVT). The optimal electroporation conditions for gene transfer into CTVTs were tested by luciferase activity and determined to be a voltage of 200 V and duration of 50 msec, with the number of shocks set at 10 pulses, and the use of an electrode with 2 needles. Under these conditions, intratumoral administration of as little as 0.1 mg pIL-12 followed by electroporation significantly inhibited the growth of well-established tumors and eventually led to complete tumor regression. Furthermore, local pIL-12 treatment also induced a strong systemic effect that prevented new tumor growth and cured established tumors at distant locations. Intratumoral administration of pIL-12 greatly elevated the IL-12 level in the tumor masses, but produced only a trace amount in the serum. A high level of IFN-gamma mRNA was also detected in the treated tumor masses. pIL-12 gene therapy attracted significantly more lymphocytes infiltrating the tumors, including CD4(+) and CD8(+) T cells, and the surface expression of MHC I and MHC II molecules on CTVT cells was greatly increased after pIL-12 therapy. This treatment also induced apoptosis of the tumor cells as detected by Annexin V. More importantly, delivery of pIL-12 with intratumoral electroporation did not result in any detectable toxicity in the dogs. We conclude that intratumoral electroporation of the pIL-12 gene could cause profound immunologic host responses and efficiently treat CTVT in beagle dogs. The results also indicate that CTVT is an excellent large animal cancer model for testing immunogene therapies mediated by electroporation.

Antitumor and antimetastatic activity of IL-23.

The structure and T cell stimulatory effects of the recently discovered cytokine IL-23 are similar to, but distinct from, those of IL-12. Although the antitumor activities of IL-12 are well characterized, the effect of IL-23 on tumor growth is not known. In this study, murine CT26 colon adenocarcinoma and B16F1 melanoma cells were engineered using retroviral vectors to release single-chain IL-23 (scIL-23) to evaluate its antitumor activity. In BALB/c mice, scIL-23-transduced CT26 cells grew progressively until day 26 to an average size of 521 +/- 333 mm(3), then the tumors started to regress in most animals, resulting in a final 70% rate of complete tumor rejection. scIL-23 transduction also significantly suppressed lung metastases of CT26 and B16F1 tumor cells. In addition, mice that rejected scIL-23-transduced tumors developed a memory response against subsequent wild-type tumor challenge. Compared with scIL-12-expressing CT26 cells, scIL-23-transduced tumors lacked the early response, but achieved comparable antitumor and antimetastatic activity. These results demonstrated that IL-23, like IL-12, provided effective protection against malignant diseases, but it probably acted by different antitumor mechanisms. As a first step in identifying these antitumor mechanisms, tumor challenge studies were performed in immunocompromised hosts and in animals selectively depleted of various lymphocyte populations. The results showed that CD8(+) T cells, but not CD4(+) T cells or NK cells, were crucial for the antitumor activity of IL-23.