Extracellular Vesicles Loaded with Long Antisense RNAs Repress Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Long antisense RNAs (asRNAs) have been observed to repress HIV and other virus expression in a manner that is refractory to viral evolution. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) disease, has a distinct ability to evolve resistance around antibody targeting, as was evident from the emergence of various SARS-CoV-2 spike antibody variants. Importantly, the effectiveness of current antivirals is waning due to the rapid emergence of new variants of concern, more recently the omicron variant. One means of avoiding the emergence of viral resistance is by using long asRNA to target SARS-CoV-2. Similar work has proven successful with HIV targeting by long asRNA. In this study, we describe a long asRNA targeting SARS-CoV-2 RNA-dependent RNA polymerase gene and the ability to deliver this RNA in extracellular vesicles (EVs) to repress virus expression. The observations presented in this study suggest that EV-delivered asRNAs are one means to targeting SARS-CoV-2 infection, which is both effective and broadly applicable as a means to control viral expression in the absence of mutation. This is the first demonstration of the use of engineered EVs to deliver long asRNA payloads for antiviral therapy.

An intranasally delivered ultra-conserved siRNA prophylactically represses SARS-CoV-2 infection in the lung and nasal cavity.

There remains a striking overall mortality burden of COVID-19 worldwide. Given the waning effectiveness of current SARS-CoV-2 antivirals due to the rapid emergence of new variants of concern (VOC), we employed a direct-acting molecular therapy approach using gene silencing RNA interference (RNAi) technology. In this study, we developed and screened several ultra-conserved small-interfering RNAs (siRNAs) before selecting one potent siRNA candidate for pre-clinical in vivo testing. This non-immunostimulatory, anti-SARS-CoV-2 siRNA candidate maintains its antiviral activity against all tested SARS-CoV-2 VOC and works effectively as a single agent. For the first time, significant antiviral effects in both the lungs and nasal cavities of SARS-CoV-2 infected mice were observed when this siRNA candidate was delivered intranasally (IN) as a prophylactic agent with the aid of lipid nanoparticles (LNPs). Importantly, a pre-exposure prophylactic IN-delivered anti-SARS-CoV-2 siRNA antiviral that can ameliorate viral replication in the nasal cavity could potentially prevent aerosol spread of respiratory viruses. An IN delivery approach would allow for the development of a direct-acting nasal spray approach that could be self-administered prophylactically.

Immune responses in children after vaccination with a typhoid Vi-tetanus toxoid conjugate vaccine in Bangladesh.

A cluster-randomized trial of Vi-TT was conducted in Dhaka, Bangladesh, using JE vaccine as the control. A subset of 1,500 children were randomly selected on 2:1 basis (Vi-TT vs JE) to assess immune response. Blood was collected before vaccination, and on days 28, 545 and 730 post-vaccination and plasma anti-Vi-IgG response was measured. A robust, persistent antibody response was induced after single dose of Vi-TT, even after 2 years of vaccination. While there is no accepted serological antibody threshold of protection, analyzing the antibodies of children who received Vi-TT provides evidence that may later be useful in predicting population protection.

The interplay between WASH practices and vaccination with oral cholera vaccines in protecting against cholera in urban Bangladesh: Reanalysis of a cluster-randomized trial.

The current global initiative to end Cholera by 2030 emphasizes the use of oral cholera vaccine (OCV) combined with feasible household Water-Sanitation-Hygiene (WASH) interventions. However, little is known about how improved WASH practices and behaviors and OCV interact to reduce the risk of cholera. We reanalyzed two arms of a cluster-randomized trial in urban Bangladesh, to evaluate the effectiveness of OCV given as a 2-dose regimen. One arm (30 clusters, n = 94,675) was randomized to vaccination of persons aged one year and older with OCV, and the other arm (30 clusters, n = 80,056) to no intervention. We evaluated the prevention of cholera by household WASH, classified at baseline using a previously validated rule, and OCV over 2 years of follow-up. When analyzed by assignment to OCV clusters rather than receipt of OCV, in comparison to persons living in "Not Better WASH" households in the control clusters, reduction of severe cholera (the primary outcome) was similar for persons in "Not Better WASH" households in vaccine clusters (46%, 95% CI:24,62), for persons in "Better WASH" households in the control clusters (48%, 95% CI:25,64), and for persons in "Better WASH" households in the vaccine clusters (48%, 95% CI:16,67). In contrast, when analyzed by actual receipt of a complete OCV regimen, , in comparison to persons in "Not Better WASH" households in the control clusters, protection against severe cholera increased steadily from 39% (95% CI:13,58) in residents of "Better WASH" households in the control clusters to 57% (95% CI:35,72) in vaccinated persons in "Not Better WASH" households to 63% (95% CI:21,83) in vaccinated persons in "Better WASH" households. This analysis suggests that improved household WASH and OCV received may interact to provide greater protection against cholera. However, the divergence between findings related to intent to vaccinate versus those pertaining to actual receipt of OCV underscores the need for further research on this topic.

Prophylactic intranasal administration of lipid nanoparticle formulated siRNAs reduce SARS-CoV-2 and RSV lung infection.

RNA interference (RNAi) is an emerging and promising therapy for a wide range of respiratory viral infections. This highly specific suppression can be achieved by the introduction of short-interfering RNA (siRNA) into mammalian systems, resulting in the effective reduction of viral load. Unfortunately, this has been hindered by the lack of a good delivery system, especially via the intranasal (IN) route. Here, we have developed an IN siRNA encapsulated lipid nanoparticle (LNP) in vivo delivery system that is highly efficient at targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory syncytial virus (RSV) lung infection in vivo. Importantly, IN siRNA delivery without the aid of LNPs abolishes anti-SARS-CoV-2 activity in vivo. Our approach using LNPs as the delivery vehicle overcomes the significant barriers seen with IN delivery of siRNA therapeutics and is a significant advancement in our ability to delivery siRNAs. The study presented here demonstrates an attractive alternate delivery strategy for the prophylactic treatment of both future and emerging respiratory viral diseases.

Hyperactivation of p53 using CRISPRa kills human papillomavirus-driven cervical cancer cells.

Clinical and pre-clinical work for a number of cancer types has demonstrated relatively positive outcomes and effective tumour regression when the level and function of p53, a well-established tumour suppressor, is restored. Human papillomavirus (HPV)-driven cancers encode the E6 oncoprotein, which leads to p53 degradation, to allow the carcinogenic process to proceed. Indeed, there have been several attempts to revive p53 function in HPV-driven cancers by both pharmacological and genetic means to increase p53 bioavailability. Here, we employed a CRISPR activation (CRISPRa) approach to overcome HPV-mediated silencing of p53 by hyperexpressing the p53 gene promoter. Our data show that CRISPRa-mediated hyperexpression of p53 leads to HPV+ cervical cancer cell killing and the reduction of cell proliferation. This proof-of-concept data suggest that increasing p53 bioavailability may potentially be a promising therapeutic approach for the treatment of HPV-driven cancers.

Analysis of a hit-and-run tumor model by HPV in oropharyngeal cancers.

Several viruses are known to be associated with the development of certain cancers, including human papilloma virus (HPV), an established causative agent for a range of anogenital and head and neck cancers. However, the causality has been based on the presence of the virus, or its genetic material, in the sampled tumors. We have long wondered if viruses cause cancer via a "hit and run" mechanism such that they are no longer present in the resulting tumors. Here, we hypothesize that the absence of viral genes from the tumor does not necessarily exclude the viral aetiology. To test this, we used an HPV-driven oropharyngeal cancer (OPC) tumor model and CRISPR to delete the viral oncogene, E7. Indeed, the genetic removal of HPV E7 oncogene eliminates tumors in vivo. Remarkably, E7 deleted tumors recurred over time and develop new mutations not previously seen in HPV+ OPC tumors. Importantly, a number of these new mutations are found to be already present in HPV- OPC tumors.

Co-administration of Oral Cholera Vaccine With Oral Polio Vaccine Among Bangladeshi Young Children: A Randomized Controlled Open Label Trial to Assess Interference.

BACKGROUND: Cholera remains a public health threat for low- and middle-income countries, particularly in Asia and Africa. Shanchol™, an inactivated oral cholera vaccine (OCV) is currently in use globally. OCV and oral poliovirus vaccines (OPV) could be administered concomitantly, but the immunogenicity and safety of coadministration among children aged 1-3 years is unknown. METHODS: We undertook an open-label, randomized, controlled, inequality trial in Dhaka city, Bangladesh. Healthy children aged 1-3 years were randomly assigned to 1 of 3 groups: bivalent OPV (bOPV)-alone, OCV-alone, or combined bOPV + OCV and received vaccines on the day of enrollment and 28 days later. Blood samples were collected on the day of enrollment, day 28, and day 56. Serum poliovirus neutralizing antibodies and vibriocidal antibodies against Vibrio cholerae O1 were assessed using microneutralization assays. RESULTS: A total of 579 children aged 1‒3 years were recruited, 193 children per group. More than 90% of the children completed visits at day 56. Few adverse events following immunization were recorded and were equivalent among study arms. On day 28, 60% (90% confidence interval: 53%-67%) and 54% (46%-61%) of participants with co-administration of bOPV + OCV responded to polioviruses type 1 and 3, respectively, compared to 55% (47%-62%) and 46% (38%-53%) in the bOPV-only group. Additionally, >50% of participants showed a ≥4-fold increase in vibriocidal antibody titer responses on day 28, comparable to the responses observed in OCV-only arm. CONCLUSIONS: Co-administration of bOPV and OCV is safe and effective in children aged 1-3 years and can be cost-beneficial. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov (NCT03581734).

Genetic deletion of HPV E7 oncogene effectively regresses HPV driven oral squamous carcinoma tumour growth.

The major HPV oncogenes, E6 and E7, are known for its notoriety in driving the carcinogenic process in human papilloma virus (HPV) driven cancers. It is well-established that the removal of E7 dampens HPV cancer cell growth and proliferation. This has made E7 an attractive target for HPV cancers. Seminal work from our laboratory employed a CRISPR editing approach to delete E7 which resulted in the effective elimination of HPV+ cervical cancer tumours in vivo. We have also successfully delayed HPV+ tumour growth in vivo with aurora kinase (AURK) inhibitors, an effect which is strongly sensitized by the presence of E7. Unlike our previous observations in cervical cancer cells, in vitro targeting of E6/E7 have only resulted in partial killing of HPV+ oral squamous carcinoma (OSC) cells. However, the effect of sustained removal of E7 on HPV+ OSC tumour growth have not been explored. In this study, we investigated a staggered combination of aurora kinase inhibition, using alisertib, followed by CRISPR editing of E7, to determine if this would lead to better HPV+ OSC killing. Remarkably, genetic deletion of E7 alone was sufficient to effectively regress established HPV+ OSC tumours in vivo suggesting that E7 is essential in the maintenance of HPV+ OSC cancers.

Nucleocapsid Specific Diagnostics for the Detection of Divergent SARS-CoV-2 Variants.

Since the start of the COVID-19 pandemic, multiple waves of SARS-CoV-2 variants have emerged. Of particular concern is the omicron variant, which harbors 28 mutations in the spike glycoprotein receptor binding and N-terminal domains relative to the ancestral strain. The high mutability of SARS-CoV-2 therefore poses significant hurdles for development of universal assays that rely on spike-specific immune detection. To address this, more conserved viral antigens need to be targeted. In this work, we comprehensively demonstrate the use of nucleocapsid (N)-specific detection across several assays using previously described nanobodies C2 and E2. We show that these nanobodies are highly sensitive and can detect divergent SARS-CoV-2 ancestral, delta and omicron variants across several assays. By comparison, spike-specific antibodies S309 and CR3022 only disparately detect SARS-CoV-2 variant targets. As such, we conclude that N-specific detection could provide a standardized universal target for detection of current and emerging SARS-CoV-2 variants of concern.

Diagnosis, Management, and Future Control of Cholera.

Cholera, caused by Vibrio cholerae, persists in developing countries due to inadequate access to safe water, sanitation, and hygiene. There are approximately 4 million cases and 143,000 deaths each year due to cholera. The disease is transmitted fecally-orally via contaminated food or water. Severe dehydrating cholera can progress to hypovolemic shock due to the rapid loss of fluids and electrolytes, which requires a rapid infusion of intravenous (i.v.) fluids. The case fatality rate exceeds 50% without proper clinical management but can be less than 1% with prompt rehydration and antibiotics. Oral cholera vaccines (OCVs) serve as a major component of an integrated control package during outbreaks or within zones of endemicity. Water, sanitation, and hygiene (WaSH); health education; and prophylactic antibiotic treatment are additional components of the prevention and control of cholera. The World Health Organization (WHO) and the Global Task Force for Cholera Control (GTFCC) have set an ambitious goal of eliminating cholera by 2030 in high-risk areas.

Hyperactivating p53 in Human Papillomavirus-Driven Cancers: A Potential Therapeutic Intervention.

Despite a vaccine being available, human papillomavirus virus (HPV)-driven cancers remain the ninth most prevalent cancers globally. Current therapies have significant drawbacks and often still lead to poor prognosis and underwhelming survival rates. With gene therapy becoming more available in the clinic, it poses a new front for therapeutic development. A characteristic of HPV-driven cancers is the ability to encode oncoproteins that aberrate normal p53 function without mutating this tumour-suppressor gene. The HPV E6 oncoprotein degrades p53 to allow the HPV-driven carcinogenic process to proceed. This review aimed to investigate the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene-editing technology and how it may be used to overcome HPV-mediated silencing of p53 by hyper-expressing the p53 promoter. Increasing p53 bioavailability may have promising potential as a therapy and has been a goal in the context of HPV-driven cancers. Clinical trials and proof-of-concept pre-clinical work have shown positive outcomes and tumour death when p53 levels are increased. Despite previous successes of RNA-based medicines, including the knockout of HPV oncogenes, the use of CRISPR activation is yet to be investigated as a promising potential therapy. This short review summarises key developments on attempts that have been made to increase p53 expression in the context of HPV cancer therapy, but leaves open the possibility for other cancers bearing a p53 wild-type gene.

RNAi to treat SARS-CoV-2-variant proofing the next generation of therapies.

There is an urgent need to bring new antivirals to SARS-CoV-2 to the market. Indeed, in the last 3 months, we have seen at least two new antivirals approved, molnupiravir and paxlovid. Both are older established antivirals that show some efficacy against SARS-CoV-2. The work by Chang et al (2022) in the current issue of EMBO Molecular Medicine explores the use of short interfering RNAs to directly target SARS-CoV-2 and shows that RNAi is an effective approach to reducing, or even eliminating viral replication, depending on the experimental setting. This antiviral effect results in significant prevention of infection-related pathology in animals. The key feature of this approach, besides its simplicity as naked siRNAs, is that all current variants are covered by this treatment.

Toward Typhoid Fever Elimination.

Salmonella enterica serotype Typhi (S Typhi) causes typhoid fever and is responsible for an estimated 9 million cases and 110,000 deaths globally per annum. Typhoid fever is endemic in areas where water, sanitation, and hygiene (WaSH) infrastructure is poor. Serious complications develop in approximately 10%-15% of patients if left untreated, and this is driven by inadequate diagnostic methods and the high burden of antibiotic-resistant strains, complicating clinical management and ultimately prognosis. Asymptomatic chronic carriers, in addition to acutely infected patients, contribute to continued transmission through the shedding of the organism in the feces. The high morbidity and mortality of typhoid fever in low- and middle-income countries reinforce the need for an integrated control approach, which may ultimately lead to elimination of the disease in the 21st century. Here we discuss the challenges faced in pursuit of typhoid fever elimination.

Engineered extracellular vesicles directed to the spike protein inhibit SARS-CoV-2.

SARS-CoV-2 (CoV-2) viral infection results in COVID-19 disease, which has caused significant morbidity and mortality worldwide. A vaccine is crucial to curtail the spread of SARS-CoV-2, while therapeutics will be required to treat ongoing and reemerging infections of SARS-CoV-2 and COVID-19 disease. There are currently no commercially available effective anti-viral therapies for COVID-19, urging the development of novel modalities. Here, we describe a molecular therapy specifically targeted to neutralize SARS-CoV-2, which consists of extracellular vesicles (EVs) containing a novel fusion tetraspanin protein, CD63, embedded within an anti-CoV-2 nanobody. These anti-CoV-2-enriched EVs bind SARS-CoV-2 spike protein at the receptor-binding domain (RBD) site and can functionally neutralize SARS-CoV-2. This work demonstrates an innovative EV-targeting platform that can be employed to target and inhibit the early stages of SARS-CoV-2 infection.

Long-term sialidase-specific immune responses after natural infection with cholera: Findings from a longitudinal cohort study in Bangladesh.

Background: Immune responses that target sialidase occur following natural cholera and have been associated with protection against cholera. Sialidase is a neuraminidase that facilitates the binding of cholera toxin (CT) to intestinal epithelial cells. Despite this, little is known about age-related sialidase-specific immune responses and the impact of nutritional status and co-infection on sialidase-specific immunity. Methods: We enrolled 50 culture-confirmed Vibrio cholerae O1 cholera cases presenting to the icddr,b Dhaka hospital with moderate to severe dehydration. We evaluated antibody responses out to 18 months (day 540) following cholera. We assessed immune responses targeting sialidase, lipopolysaccharide (LPS), cholera toxin B subunit (CtxB), and vibriocidal responses. We also explored the association of sialidase-specific immune responses to nutritional parameters and parasitic co-infection of cases. Results: This longitudinal cohort study showed age-dependent differences in anti-sialidase immune response after natural cholera infection. Adult patients developed plasma anti-sialidase IgA and IgG responses after acute infection (P<0.05), which gradually decreased from day 30 on. In children, no significant anti-sialidase IgA, IgM, and IgG response was seen with the exception of a late IgG response at study day 540 (p=0.05 compared to adults). There was a correlation between anti-sialidase IgA with vibriocidal titers, as well as anti-sialidase IgA and IgG with anti-LPS and anti-CtxB antibody responses in adult patients, whereas in children, a significant positive correlation was seen only between anti-sialidase IgA and CtxB IgA responses. Stunted children showed significantly lower anti-sialidase IgA, IgG, and IgM antibody responses and higher LPS IgG and IgM antibody responses than healthy children. The anti-sialidase IgA and IgG responses were significantly higher in cases with concomitant parasitic infection. Conclusion: Our data suggest that cholera patients develop age-distinct systemic and mucosal immune responses against sialidase. The stunted children have a lower anti-sialidase antibody response which may be associated with gut enteropathy and the neuraminidase plays an important role in augmented immune response in cholera patients infected with parasites.

Complete protection by a single-dose skin patch-delivered SARS-CoV-2 spike vaccine.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 160 million people and resulted in more than 3.3 million deaths, and despite the availability of multiple vaccines, the world still faces many challenges with their rollout. Here, we use the high-density microarray patch (HD-MAP) to deliver a SARS-CoV-2 spike subunit vaccine directly to the skin. We show that the vaccine is thermostable on the patches, with patch delivery enhancing both cellular and antibody immune responses. Elicited antibodies potently neutralize clinically relevant isolates including the Alpha and Beta variants. Last, a single dose of HD-MAP­delivered spike provided complete protection from a lethal virus challenge in an ACE2-transgenic mouse model. Collectively, these data show that HD-MAP delivery of a SARS-CoV-2 vaccine was superior to traditional needle-and-syringe vaccination and may be a significant addition to the ongoing COVID-19 (coronavirus disease 2019) pandemic.

A SARS-CoV-2 targeted siRNA-nanoparticle therapy for COVID-19.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. Despite several emerging vaccines, there remains no verifiable therapeutic targeted specifically to the virus. Here we present a highly effective small interfering RNA (siRNA) therapeutic against SARS-CoV-2 infection using a novel lipid nanoparticle (LNP) delivery system. Multiple siRNAs targeting highly conserved regions of the SARS-CoV-2 virus were screened, and three candidate siRNAs emerged that effectively inhibit the virus by greater than 90% either alone or in combination with one another. We simultaneously developed and screened two novel LNP formulations for the delivery of these candidate siRNA therapeutics to the lungs, an organ that incurs immense damage during SARS-CoV-2 infection. Encapsulation of siRNAs in these LNPs followed by in vivo injection demonstrated robust repression of virus in the lungs and a pronounced survival advantage to the treated mice. Our LNP-siRNA approaches are scalable and can be administered upon the first sign of SARS-CoV-2 infection in humans. We suggest that an siRNA-LNP therapeutic approach could prove highly useful in treating COVID-19 disease as an adjunctive therapy to current vaccine strategies.