The fentanyl-specific antibody FenAb024 can shield against carfentanil effects.

The surge in opioid-related deaths, driven predominantly by fentanyl and its synthetic derivatives, has become a critical public health concern, which is particularly evident in the United States. While the situation is less severe in Europe, the European Monitoring Centre for Drugs and Drug Addiction reports a rise in drug overdose deaths, with emerging concerns about the impact of fentanyl-related molecules. Synthetic opioids, initially designed for medical use, have infiltrated illicit markets due to their low production costs and high potency, with carfentanil posing additional threats, including potential chemical weaponization. Existing overdose mitigation heavily relies on naloxone, requiring timely intervention and caregiver presence, while therapeutic prevention strategies face many access challenges. To provide an additional treatment option, we propose the use of a fentanyl-specific monoclonal antibody (mAb), as a non-opioid method of prophylaxis against fentanyl and carfentanil. This mAb shows protection from opioid effects in a pre-clinical murine model. mAbs could emerge as a versatile countermeasure in civilian and biodefense settings, offering a novel approach to combat opioid-associated mortality.

A trypanosome-derived immunotherapeutics platform elicits potent high-affinity antibodies, negating the effects of the synthetic opioid fentanyl.

Poorly immunogenic small molecules pose challenges for the production of clinically efficacious vaccines and antibodies. To address this, we generate an immunization platform derived from the immunogenic surface coat of the African trypanosome. Through sortase-based conjugation of the target molecules to the variant surface glycoprotein (VSG) of the trypanosome surface coat, we develop VSG-immunogen array by sortase tagging (VAST). VAST elicits antigen-specific memory B cells and antibodies in a murine model after deploying the poorly immunogenic molecule fentanyl as a proof of concept. We also develop a single-cell RNA sequencing (RNA-seq)-based computational method that synergizes with VAST to specifically identify memory B cell-encoded antibodies. All computationally selected antibodies bind to fentanyl with picomolar affinity. Moreover, these antibodies protect mice from fentanyl effects after passive immunization, demonstrating the ability of these two coupled technologies to elicit therapeutic antibodies to challenging immunogens.

Recurrent Frameshift Neoantigen Vaccine Elicits Protective Immunity With Reduced Tumor Burden and Improved Overall Survival in a Lynch Syndrome Mouse Model.

BACKGROUND & AIMS: DNA mismatch repair deficiency drives microsatellite instability (MSI). Cells with MSI accumulate numerous frameshift mutations. Frameshift mutations affecting cancer-related genes may promote tumorigenesis and, therefore, are shared among independently arising MSI tumors. Consequently, such recurrent frameshift mutations can give rise to shared immunogenic frameshift peptides (FSPs) that represent ideal candidates for a vaccine against MSI cancer. Pathogenic germline variants of mismatch repair genes cause Lynch syndrome (LS), a hereditary cancer syndrome affecting approximately 20-25 million individuals worldwide. Individuals with LS are at high risk of developing MSI cancer. Previously, we demonstrated safety and immunogenicity of an FSP-based vaccine in a phase I/IIa clinical trial in patients with a history of MSI colorectal cancer. However, the cancer-preventive effect of FSP vaccination in the scenario of LS has not yet been demonstrated. METHODS: A genome-wide database of 488,235 mouse coding mononucleotide repeats was established, from which a set of candidates was selected based on repeat length, gene expression, and mutation frequency. In silico prediction, in vivo immunogenicity testing, and epitope mapping was used to identify candidates for FSP vaccination. RESULTS: We identified 4 shared FSP neoantigens (Nacad [FSP-1], Maz [FSP-1], Senp6 [FSP-1], Xirp1 [FSP-1]) that induced CD4/CD8 T cell responses in naïve C57BL/6 mice. Using VCMsh2 mice, which have a conditional knockout of Msh2 in the intestinal tract and develop intestinal cancer, we showed vaccination with a combination of only 4 FSPs significantly increased FSP-specific adaptive immunity, reduced intestinal tumor burden, and prolonged overall survival. Combination of FSP vaccination with daily naproxen treatment potentiated immune response, delayed tumor growth, and prolonged survival even more effectively than FSP vaccination alone. CONCLUSIONS: Our preclinical findings support a clinical strategy of recurrent FSP neoantigen vaccination for LS cancer immunoprevention.

NMD inhibition by 5-azacytidine augments presentation of immunogenic frameshift-derived neoepitopes.

Frameshifted protein sequences elicit tumor-specific T cell-mediated immune responses in microsatellite-unstable (MSI) cancers if presented by HLA class I molecules. However, their expression and presentation are limited by nonsense-mediated RNA decay (NMD). We employed an unbiased immunopeptidomics workflow to analyze MSI HCT-116 cells and identified >10,000 HLA class I-presented peptides including five frameshift-derived InDel neoepitopes. Notably, pharmacological NMD inhibition with 5-azacytidine stabilizes frameshift-bearing transcripts and increases the HLA class I-mediated presentation of InDel neoepitopes. The frameshift mutation underlying one of the identified InDel neoepitopes is highly recurrent in MSI colorectal cancer cell lines and primary patient samples, and immunization with the corresponding neoepitope induces strong CD8+ T cell responses in an HLA-A∗02:01 transgenic mouse model. Our data show directly that pharmacological NMD inhibition augments HLA class I-mediated presentation of immunogenic frameshift-derived InDel neoepitopes thus highlighting the clinical potential of NMD inhibition in anti-cancer immunotherapy strategies.

The shared frameshift mutation landscape of microsatellite-unstable cancers suggests immunoediting during tumor evolution.

The immune system can recognize and attack cancer cells, especially those with a high load of mutation-induced neoantigens. Such neoantigens are abundant in DNA mismatch repair (MMR)-deficient, microsatellite-unstable (MSI) cancers. MMR deficiency leads to insertion/deletion (indel) mutations at coding microsatellites (cMS) and to neoantigen-inducing translational frameshifts. Here, we develop a tool to quantify frameshift mutations in MSI colorectal and endometrial cancer. Our results show that frameshift mutation frequency is negatively correlated to the predicted immunogenicity of the resulting peptides, suggesting counterselection of cell clones with highly immunogenic frameshift peptides. This correlation is absent in tumors with Beta-2-microglobulin mutations, and HLA-A*02:01 status is related to cMS mutation patterns. Importantly, certain outlier mutations are common in MSI cancers despite being related to frameshift peptides with functionally confirmed immunogenicity, suggesting a possible driver role during MSI tumor evolution. Neoantigens resulting from shared mutations represent promising vaccine candidates for prevention of MSI cancers.

Macrophage-derived nitric oxide initiates T-cell diapedesis and tumor rejection.

In tumor biology, nitric oxide (NO) is generally regarded as an immunosuppressive molecule that impedes T-cell functions and activation of endothelial cells. Contrasting with this view, we here describe a critical role for NO derived from inducible nitric oxide (iNOS)-expressing tumor macrophages in T-cell infiltration and tumor rejection as shown by iNOS gene deletion, inhibition of iNOS, or NO donors. Specifically, macrophage-derived NO was found to induce on tumor vessels adhesion molecules that were required for T-cell extravasation. Experiments with human endothelial cells revealed a bimodal dose-dependent effect of NO. High doses of NO donors were indeed suppressive but lower, more physiological concentrations, induced adhesion molecules in an NFkB-dependent pathway and preferentially activated transcription of genes involved in lymphocyte diapedesis. iNOS+ macrophages in tumors appear to generate precisely the amount of NO that promotes endothelial activation and T-cell infiltration. These results will be valuable for the development of strategies designed to overcome the paucity of T-cell infiltration into tumors that is a major obstacle in clinical cancer immunotherapy.

Segregation of late outgrowth endothelial cells into functional endothelial CD34- and progenitor-like CD34+ cell populations.

Late outgrowth endothelial cells (OECs) that originate from peripheral blood mononuclear cells ex vivo have phenotypic and functional properties of mature endothelial cells. Given the potential therapeutic applications of OECs, understanding their biology is crucial. We have identified two distinct OEC populations based on differential expression of the cell surface marker CD34. OEC colonies lacked CD34 expression (CD34-), expressed CD34 in the majority of cells (CD34+), or showed a mixed expression pattern within a colony (CD34+/-). CD34+ and CD34- OECs were negative for hematopoietic cell marker CD45 and expressed the endothelial cell surface markers CD31, CD146, CD105, and VEGFR-2. Functionally CD34- and CD34+ OECs exhibited strikingly distinct behaviors. CD34- OECs, unlike CD34+ OECs, were capable of sprouting, formed tubes, and responded to angiogenic growth factors in vitro. In vivo, CD34- OECs formed endothelial tubes, while CD34+ OECs, despite being unable to form tubes, promoted infiltration of murine vasculature. Global gene expression profiling in CD34- and CD34+ OECs identified functional importance of the MMP-1/PAR-1 pathway in CD34- OECs. MMP-1 stimulated the expression of VEGFR-2, neuropilin-1, neuropilin-2, and CXCR4 and activated ERK1/2, whereas down-regulation of PAR-1 in CD34- OECs resulted in impaired angiogenic responses in vitro and reduced VEGFR-2 levels. In contrast, the CD34+ OEC colonies expressed high levels of the progenitor cell marker ALDH, which was absent in CD34- OECs. In summary, we show that OECs can be classified into functionally mature endothelial cells (CD34- OECs) that depend on the MMP-1/PAR-1 pathway and progenitor-like angiogenesis-promoting cells (CD34+ OECs).

Descriptive characterization of the 2010 cholera outbreak in Nigeria.

BACKGROUND: In 2010, 18 States of Nigeria reported cholera outbreaks with a total of 41,787 cases including 1,716 deaths (case-fatality rate [CFR]: 4.1%). This exceeded the mean overall CFR of 2.4% reported in Africa from 2000-2005 and the WHO acceptable rate of 1%. We conducted a descriptive analysis of the 2010 cholera outbreak to determine its epidemiological and spatio-temporal characteristics. METHODS: We conducted retrospective analysis of line lists obtained from 10 of the 18 states that submitted line lists to the Federal Ministry of Health (FMOH). We described the outbreak by time, place and person and calculated the attack rates by state as well as the age- and sex-specific CFR from cholera cases for whom information on age, sex, place of residence, onset of symptoms and outcome were available. RESULTS: A total of 21,111 cases were reported with an overall attack rate and CFR of 47.8 cases /100,000 population and 5.1%, respectively. The CFR ranged in the states between 3.8% and 8.9%. The age-specific CFR was highest among individuals 65 years and above (14.6%). The epidemiological curve showed three peaks with increasing number of weekly reported cases. A geographical clustering of LGAs reporting cholera cases could be seen in all ten states. During the third peak which coincided with flooding in five states the majority of newly affected LGAs were situated next to LGAs with previously reported cholera cases, only few isolated outbreaks were seen. CONCLUSION: Our study showed a cholera outbreak that grew in magnitude and spread to involve the whole northern part of the country. It also highlights challenges of suboptimal surveillance and response in developing countries as well as potential endemicity of cholera in the northern part of Nigeria. There is the need for a harmonized, coordinated approach to cholera outbreaks through effective surveillance and response with emphasis on training and motivating front line health workers towards timely detection, reporting and response. Findings from the report should be interpreted with caution due to the high number of cases with incomplete information, and lack of data from eight states.

No evidence of oncogenic KRAS mutations in squamous cell carcinomas of the anogenital tract and head and neck region independent of human papillomavirus and p16(INK4a) status.

Carcinogenesis of squamous cell carcinomas (SCCs) in the anogenital tract and head and neck region is heterogeneous. A substantial proportion of SCC in the vulva, anus, and head and neck follows a human papillomavirus (HPV)-induced carcinogenic pathway. However, the molecular pathways of carcinogenesis in the HPV-independent lesions are not completely understood. We hypothesized that oncogenic Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations might represent a carcinogenic mechanism in a proportion of those HPV-negative cancers. Considering the repeated observation of KRAS-associated p16(INK4a) overexpression in human tumors, it was assumed that KRAS mutations might be particularly present in the group of HPV-negative, p16(INK4a)-positive cancers. To test this hypothesis, we analyzed 66 anal, vulvar, and head and neck SCC with known immunohistochemical p16(INK4a) and HPV DNA status for KRAS mutations in exon 2 (codons 12, 13, and 15). We enriched the tumor collection with HPV DNA-negative, p16(INK4a)-positive cancers. A subset of 37 cancers was also analyzed for mutations in the B-Raf proto-oncogene, serine/threonine kinase (BRAF) gene. None of the 66 tumors harbored mutations in KRAS exon 2, thus excluding KRAS mutations as a common event in SCC of the anogenital and head and neck region and as a cause of p16(INK4a) expression in these tumors. In addition, no BRAF mutations were detected in the 37 analyzed tumors. Further studies are required to determine the molecular events underlying HPV-negative anal, vulvar, and head and neck carcinogenesis. Considering HPV-independent p16(INK4a) overexpression in some of these tumors, particular focus should be placed on alternative upstream activators and potential downstream disruption of the p16(INK4a) pathway.