Prospects and Challenges in Developing mRNA Vaccines for Infectious Diseases and Oncogenic Viruses.

mRNA vaccines have emerged as an optimistic technological platform for vaccine innovation in this new scientific era. mRNA vaccines have dramatically altered the domain of vaccinology by offering a versatile and rapid approach to combating infectious diseases and virus-induced cancers. Clinical trials have demonstrated efficacy rates of 94-95% in preventing COVID-19, and mRNA vaccines have been increasingly recognized as a powerful vaccine platform. Although mRNA vaccines have played an essential role in the COVID-19 pandemic, they still have several limitations; their instability and degradation affect their storage, delivery, and over-all efficiency. mRNA is typically enclosed in a transport mechanism to facilitate its entry into the target cell because it is an unstable and negatively charged molecule. For instance, mRNA that is given using lipid-nanoparticle-based vaccine delivery systems (LNPs) solely enters cells through endocytosis, establishing an endosome without damaging the cell membrane. The COVID-19 pandemic has accelerated the development of mRNA vaccine platforms used to treat and prevent several infectious diseases. This technology has the potential to change the future course of the disease by providing a safe and effective way to combat infectious diseases and cancer. A single-stranded genetic sequence found in mRNA vaccines instructs host cells to produce proteins inside ribosomes to elicit immunological responses and prepare the immune system to fight infections or cancer cells. The potential applications of mRNA vaccine technology are vast and can lead to the development of a preferred vaccine pattern. As a result, a new generation of vaccinations has gradually gained popularity and access to the general population. To adapt the design of an antigen, and even combine sequences from different variations in response to new changes in the viral genome, mRNA vaccines may be used. Current mRNA vaccines provide adequate safety and protection, but the duration of that protection can only be determined if further clinical research is conducted.

A measles-vectored vaccine candidate expressing prefusion-stabilized SARS-CoV-2 spike protein brought to phase I/II clinical trials: protection of African green monkeys from COVID-19 disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and is responsible for the largest human pandemic in 100 years. Thirty-four vaccines are currently approved for use worldwide, and approximately 67% of the world population has received a complete primary series of one, yet countries are dealing with new waves of infections, variant viruses continue to emerge, and breakthrough infections are frequent secondary to waning immunity. Here, we evaluate a measles virus (MV)-vectored vaccine expressing a stabilized prefusion SARS-CoV-2 spike (S) protein (MV-ATU3-S2PΔF2A; V591) with demonstrated immunogenicity in mouse models (see companion article [J. Brunet, Z. Choucha, M. Gransagne, H. Tabbal, M.-W. Ku et al., J Virol 98:e01693-23, 2024, https://doi.org/10.1128/jvi.01693-23]) in an established African green monkey model of disease. Animals were vaccinated with V591 or the control vaccine (an equivalent MV-vectored vaccine with an irrelevant antigen) intramuscularly using a prime/boost schedule, followed by challenge with an early pandemic isolate of SARS-CoV-2 at 56 days post-vaccination. Pre-challenge, only V591-vaccinated animals developed S-specific antibodies that had virus-neutralizing activity as well as S-specific T cells. Following the challenge, V591-vaccinated animals had lower infectious virus and viral (v) RNA loads in mucosal secretions and stopped shedding virus in these secretions earlier. vRNA loads were lower in these animals in respiratory and gastrointestinal tract tissues at necropsy. This correlated with a lower disease burden in the lungs as quantified by PET/CT at early and late time points post-challenge and by pathological analysis at necropsy.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the largest human pandemic in 100 years. Even though vaccines are currently available, countries are dealing with new waves of infections, variant viruses continue to emerge, breakthrough infections are frequent, and vaccine hesitancy persists. This study uses a safe and effective measles vaccine as a platform for vaccination against SARS-CoV-2. The candidate vaccine was used to vaccinate African green monkeys (AGMs). All vaccinated AGMs developed robust antigen-specific immune responses. After challenge, these AGMs produced less virus in mucosal secretions, for a shorter period, and had a reduced disease burden in the lungs compared to control animals. At necropsy, lower levels of viral RNA were detected in tissue samples from vaccinated animals, and the lungs of these animals lacked the histologic hallmarks of SARS-CoV-2 disease observed exclusively in the control AGMs.

Review: N1-methyl-pseudouridine (m1Ψ): Friend or foe of cancer?

Due to the health emergency created by SARS-CoV-2, the virus that causes the COVID-19 disease, the rapid implementation of a new vaccine technology was necessary. mRNA vaccines, being one of the cutting-edge new technologies, attracted significant interest and offered a lot of hope. The potential of these vaccines in preventing admission to hospitals and serious illness in people with comorbidities has recently been called into question due to the vaccines' rapidly waning immunity. Mounting evidence indicates that these vaccines, like many others, do not generate sterilizing immunity, leaving people vulnerable to recurrent infections. Additionally, it has been discovered that the mRNA vaccines inhibit essential immunological pathways, thus impairing early interferon signaling. Within the framework of COVID-19 vaccination, this inhibition ensures an appropriate spike protein synthesis and a reduced immune activation. Evidence is provided that adding 100 % of N1-methyl-pseudouridine (m1Ψ) to the mRNA vaccine in a melanoma model stimulated cancer growth and metastasis, while non-modified mRNA vaccines induced opposite results, thus suggesting that COVID-19 mRNA vaccines could aid cancer development. Based on this compelling evidence, we suggest that future clinical trials for cancers or infectious diseases should not use mRNA vaccines with a 100 % m1Ψ modification, but rather ones with the lower percentage of m1Ψ modification to avoid immune suppression.

Re-inventing protection in a post-pandemic world: A new aerodynamic endonasal filtration technology.

Is there a "missing device" for respiratory personal protection? Does it exist an easy-to-use device, allowing extensive use in everyday settings by the population, maximizing tolerability and low visual and physical invasiveness protecting from a wide range of threats including airborne pathogens, hence including the particle range of fine and ultrafine particles? Looking at the recent past, in the urgency of finding ready-to-use solutions for the respiratory protection of the population during the outbreak of the SARS-CoV-2 pandemic, devices for occupational safety have been used, such as filtering face masks. These are devices intended for workers operating during work shifts in environments characterized by potential high risk, known a priori, often directly sensible; this makes wearers motivated to tolerate discomfort for a given period to face a localized risk, and safety managers determined to supervise compliance with usage specifications. Their use by general population has implied known shortcomings, such as weak compatibility with relational work and activities, low tolerability during prolonged use, low compliance with the proper use of the device, all of this lessening actual protection. The need for a new perspective has emerged, targeting effectiveness in whole daily life, rather than punctual efficacy. Nasal filters are promising candidates to protect individuals throughout the day during the most varied activities, but they lack a systematic definition as a device and as a product; it follows that the high complexity needed to reach an effective performance envelop is generally underestimated. By reviewing available literature, the present paper draws on the experience from the pandemic and infers systematic product specifications and characterization methods for a new, effective personal respiratory protection device; these specifications are compared with the stringent constraints associated with the endonasal applications and, based on air filtration state of the art, quantifies the need for technology disruption and outlining possible new development paths.

Pathogen evolution, prevention/control strategy and clinical features of COVID-19: experiences from China.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported at the end of 2019 as a worldwide health concern causing a pandemic of unusual viral pneumonia and many other organ damages, which was defined by the World Health Organization as coronavirus disease 2019 (COVID-19). The pandemic is considered a significant threat to global public health till now. In this review, we have summarized the lessons learnt during the emergence and spread of SARS-CoV-2, including its prototype and variants. The overall clinical features of variants of concern (VOC), heterogeneity in the clinical manifestations, radiology and pathology of COVID-19 patients are also discussed, along with advances in therapeutic agents.

[Surgical treatment of COVID-19 patients during the pandemic]. / Opyt khirurgicheskogo lecheniya patsientov s COVID-19 v period pandemii.

OBJECTIVE: To analyze the results of emergency surgery in COVID-19 patients with viral pneumonia. MATERIAL AND METHODS: A retrospective study included 75 COVID-19 patients who underwent emergency surgical interventions. Comorbidities included cardiac diseases, nonspecific lung diseases, type 2 diabetes, kidney diseases, overweight, and cancer. Various combinations of these diseases were also noted. RESULTS: We carried out emergency surgeries for abdominal, thoracic, soft tissue and venous diseases. Postoperative mortality was 42.6%. The best results were obtained after minimally invasive interventions without mechanical ventilation. Extended surgery with mechanical ventilation was followed by fast progression of pneumonia according to clinical and CT data. CONCLUSION: Surgical interventions undoubtedly worsen prognosis of treatment in patients with COVID-19. Emergency minimally invasive surgery without mechanical ventilation can reduce the risk of unfavorable outcomes in patients with viral pneumonia, especially in case of concomitant cancer and other severe comorbidities.

Fall of viral and bacterial pneumonia hospitalizations following COVID-19 pandemic mitigation strategies: a central Italian Region retrospective study.

Community-Acquired Pneumonia (CAP) represents one of the first causes of hospitalization and death in the elderly all over the world and weighs heavily on public health system. Since the beginning of the COVID-19 (CoronaVirus Disease-19) pandemic, everybody's behavior was forced to change, as the result of a global lockdown strategy and the obligation of using personal protection equipment (PPE). We aimed to evaluate how the mitigation strategies adopted to fight SARS-CoV-2 (Severe Acute Respiratory Coronavirus Syndrome 2) infection have influenced hospitalizations due to CAP in two different Local Health Boards (LHBs) of central Italy. We considered two main periods of observation: before and after the national start of lockdown, in two Abruzzo's LHBs. We analyzed 19,558 hospital discharge records of bacterial and viral CAP. Excluding SARS-CoV2 infection, a significant decrease in CAP hospitalizations was observed. Through the analysis of Diagnosis Related Group (DRG) values, we highlighted a significant saving of founds for the Regional Health Service. The enactment of social distancing measures to contain COVID-19 spread, brought down admissions for bacterial and viral pneumonia. Our study emphasizes that costs for hospitalizations due to CAP could be drastically reduced by mask wearing and social distancing.

Human coronaviruses: The emergence of SARS-CoV-2 and management of COVID-19.

To date, a total of seven human coronaviruses (HCoVs) have been identified, all of which are important respiratory pathogens. Recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has led to a global pandemic causing millions of infections and deaths. Here, we summarize the discovery and fundamental virology of HCoVs, discuss their zoonotic transmission and highlight the weak species barrier of SARS-CoV-2. We also discuss the possible origins of SARS-CoV-2 variants of concern identified to date and discuss the experimental challenges in characterizing mutations of interest and propose methods to circumvent them. As the COVID-19 treatment and prevention landscape rapidly evolves, we summarize current therapeutics and vaccines, and their implications on SARS-CoV-2 variants. Finally, we explore how interspecies transmission of SARS-CoV-2 may drive the emergence of novel strains, how disease severity may evolve and how COVID-19 will likely continue to burden healthcare systems globally.

Prophylactic use of hydroxychloroquine (HCQS) in COVID-19: A questionnaire-based study in health care professionals.

Background: Coronavirus disease-2019 (COVID-19) pandemic has engulfed the whole world with millions getting infected and losing their lives. Health care professionals (HCPs) who are in the front line of fighting with COVID-19 are particularly vulnerable and it is crucial to protect them from COVID-19. In this regard, the Indian Council of Medical Research (ICMR) has recommended hydroxychloroquine (HCQS) chemoprophylaxis in HCPs. However, considering the lack of sufficient evidence the HCP are in a dilemma about this aspect. Moreover, there is a paucity of data on use of HCQS as a chemoprophylaxis among Indian HCP. Hence, this study was carried out to study the extent of use and also the perception of Indian HCP toward use of HCQS as a chemoprophylaxis for COVID-19. Materials and Methods: This was a cross-sectional study done on 205 HCPs working across India. The responses were collected electronically using a prevalidated semi-structured questionnaire. Results: 62.9% (129/205) respondents reported having taken HCQS chemo-prophylaxis for COVID-19 while 34.7% (76/205) did not take it. Among HCP, who did not take the prophylaxis, the main reasons cited were concern about adverse effects (61.5%) and lack of robust evidence (24%). Only 14% of respondents felt that there was sufficient evidence to justify use of HCQS for prophylaxis while an overwhelming majority (86%) felt otherwise or were uncertain. Conclusion: The majority of participants felt that despite a lack of proven efficacy, ICMR guidelines on HCQS prophylaxis in COVID-19 are justified considering the pandemic situation. Our study also found that HCQS is well tolerated by participants and there was no reported serious adverse effect and cardiac-related side effects among them.

RésuméContexte: La pandémie de maladie à coronavirus-2019 (COVID-19) a englouti le monde entier avec des millions de personnes infectées et perdant leur des vies. Les professionnels de la santé (HCP) qui sont en première ligne dans la lutte contre le COVID-19 sont particulièrement vulnérables et il est essentiel de les protéger du COVID-19. À cet égard, le Conseil indien de la recherche médicale (ICMR) a recommandé l'hydroxychloroquine (HCQS) chimioprophylaxie chez les professionnels de la santé. Cependant, compte tenu du manque de preuves suffisantes, le professionnel de la santé est confronté à un dilemme à propos de cet aspect. En outre, il y a peu de données sur l'utilisation du HCQS comme chimioprophylaxie parmi les professionnels de la santé indiens. Par conséquent, cette étude a été menée pour étudier l'étendue de l'utilisation et aussi la perception des professionnels de la santé indiens à l'égard de l'utilisation du HCQS comme chimioprophylaxie pour le COVID-19. Matériel et Méthodes: c'était un étude transversale réalisée sur 205 professionnels de la santé travaillant en Inde. Les réponses ont été recueillies par voie électronique à l'aide d'un système semi-structuré prévalidé questionnaire. Résultats: 62,9% (129/205) des répondants ont déclaré avoir suivi une chimioprophylaxie HCQS pour le COVID-19, tandis que 34,7% (76/205) ne l'a pas pris. Parmi les professionnels de la santé qui n'ont pas pris de prophylaxie, les principales raisons invoquées étaient la préoccupation concernant les effets indésirables (61,5%) et le manque de preuves solides (24%). Seulement 14% des répondants estimaient qu'il y avait des preuves suffisantes pour justifier l'utilisation du HCQS à des fins prophylactiques alors qu'un une écrasante majorité (86%) pensait le contraire ou était incertaine. Conclusion: La majorité des participants ont estimé que malgré un manque de efficacité, les lignes directrices de l'ICMR sur la prophylaxie du HCQS dans le COVID-19 sont justifiées compte tenu de la situation pandémique. Notre étude a également révélé que Le HCQS est bien toléré par les participants et aucun effet indésirable grave ni effet secondaire cardiaque n'a été signalé parmi eux. Mots-clés: Chimioprophylaxie, COVID-19, hydroxychloroquine, Conseil indien de la recherche médicale.

Management of a Nursing Unit in a Temporary COVID-19 Specialized Hospital in Wuhan, China.

The purpose of this article was to summarize the experience of conversion and management of a nursing unit in a newly revised coronavirus disease 2019 (COVID-19) specialized hospital during the outbreak of COVID-19 in Wuhan, China. Six characteristics of management were included: nurse selection and training, transformation of ward layout, nurse position setting, quality control, humanistic care, and safety and comfort of individual protection. Orderly and efficient nursing management during COVID-19 treatment is very important to ensure the quality of clinical nursing, improve the cure rate and avoid the infection of nurses. This practical experience of the establishment and management of the nursing unit can provide reference for the nursing management of other public health events, such as the treatment of infectious diseases.

Artificial Intelligence against COVID-19 Pandemic: A Comprehensive Insight.

COVID-19 is a pandemic initially identified in Wuhan, China, which is caused by a novel coronavirus, also recognized as the Severe Acute Respiratory Syndrome (SARS-nCoV-2). Unlike other coronaviruses, this novel pathogen may cause unusual contagious pain, which results in viral pneumonia, serious heart problems, and even death. Researchers worldwide are continuously striving to develop a cure for this highly infectious disease, yet there are no well-defined absolute treatments available at present. Several vaccination drives using emergency use authorisation vaccines have been held across many countries; however, their long-term efficacy and side-effects studies are yet to be studied. Various analytical and statistical models have been developed, however, their outcome rate is prolonged. Thus, modern science stresses the application of state-of-the-art methods to combat COVID-19. This paper aims to provide a deep insight into the comprehensive literature about AI and AI-driven tools in the battle against the COVID-19 pandemic. The high efficacy of these AI systems can be observed in terms of highly accurate results, i.e., > 95%, as reported in various studies. The extensive literature reviewed in this paper is divided into five sections, each describing the application of AI against COVID-19 viz. COVID-19 prevention, diagnostic, infection spread trend prediction, therapeutic and drug repurposing. The application of Artificial Intelligence (AI) and AI-driven tools are proving to be useful in managing and fighting against the COVID-19 pandemic, especially by analysing the X-Ray and CT-Scan imaging data of infected subjects, infection trend predictions, etc.

COVID-19 Restrictions Are Associated With a Significant Decrease of All Common Respiratory Viral Illnesses in Children.

To combat the spread of coronavirus disease 2019 (COVID-19), significant measures were enacted including school and business closures, social distancing, and facial coverings. We hypothesized that this would have an impact on all respiratory infections in children. Using nasopharyngeal panel test results of children in the emergency department, we evaluated cross-sectional data from February to May in both 2019 and 2020. Respiratory panel testing included 11 common respiratory viruses and bacteria. After the restrictions were enacted, we observed a large drop in the number and percentage positive of all common respiratory viral infections in 2020 compared with the same time in 2019. When analyzing data from children <2 years old, a similar decrease was seen. Restrictions enacted to prevent the spread of COVID-19 were associated with a significant decrease in respiratory viral infections in children of all ages. This association could guide future public health recommendations and guidelines.

CCR2 Signaling Restricts SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a historic pandemic of respiratory disease (coronavirus disease 2019 [COVID-19]), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2 signaling restricts the viral burden in the lung. We find that a recently developed mouse-adapted SARS-CoV-2 (MA-SARS-CoV-2) strain as well as the emerging B.1.351 variant trigger an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using intravital antibody labeling, we demonstrate that MA-SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. Single-cell RNA sequencing (scRNA-Seq) analysis of lung homogenates identified a hyperinflammatory monocyte profile. We utilize this model to demonstrate that mechanistically, CCR2 signaling promotes the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified a potential CCR2-monocyte axis that is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 has caused a historic pandemic of respiratory disease (COVID-19), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2-dependent infiltration of monocytes restricts the viral burden in the lung. We find that SARS-CoV-2 triggers an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using RNA sequencing and flow cytometry approaches, we demonstrate that SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. Mechanistically, CCR2 signaling promoted the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified that the CCR2 pathway is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection.

Impact of Vaccination in the Rate of COVID-19 Staff Infection in an Acute Inpatient Rehabilitation Facility.

ABSTRACT: This brief report summarizes the comparative experience of an inpatient rehabilitation facility dealing with two episodes of COVID-19 infection, one before and one after the availability of vaccination, which was deployed to staff. The experience exemplifies the high rate of infection and potential for asymptomatic presentation of COVID-19 as well as the protective advantage of the vaccine for healthcare workers in this report. With a significant reduction in the rate of infection, from nearly 30% before vaccination to only 2.5% after vaccination. The data presented should serve as an encouragement for vaccination across all populations.

Protectins PCTR1 and PD1 Reduce Viral Load and Lung Inflammation During Respiratory Syncytial Virus Infection in Mice.

Viral pneumonias are a major cause of morbidity and mortality, owing in part to dysregulated excessive lung inflammation, and therapies to modulate host responses to viral lung injury are urgently needed. Protectin conjugates in tissue regeneration 1 (PCTR1) and protectin D1 (PD1) are specialized pro-resolving mediators (SPMs) whose roles in viral pneumonia are of interest. In a mouse model of Respiratory Syncytial Virus (RSV) pneumonia, intranasal PCTR1 and PD1 each decreased RSV genomic viral load in lung tissue when given after RSV infection. Concurrent with enhanced viral clearance, PCTR1 administration post-infection, decreased eosinophils, neutrophils, and NK cells, including NKG2D+ activated NK cells, in the lung. Intranasal PD1 administration post-infection decreased lung eosinophils and Il-13 expression. PCTR1 increased lung expression of cathelicidin anti-microbial peptide and decreased interferon-gamma production by lung CD4+ T cells. PCTR1 and PD1 each increased interferon-lambda expression in human bronchial epithelial cells in vitro and attenuated RSV-induced suppression of interferon-lambda in mouse lung in vivo. Liquid chromatography coupled with tandem mass spectrometry of RSV-infected and untreated mouse lungs demonstrated endogenous PCTR1 and PD1 that decreased early in the time course while cysteinyl-leukotrienes (cys-LTs) increased during early infection. As RSV infection resolved, PCTR1 and PD1 increased and cys-LTs decreased to pre-infection levels. Together, these results indicate that PCTR1 and PD1 are each regulated during RSV pneumonia, with overlapping and distinct mechanisms for PCTR1 and PD1 during the resolution of viral infection and its associated inflammation.

Tobacco use, tuberculosis and Covid-19: A lethal triad.

Smoking, TB and Covid-19 are high prevalence entities with public health consequences. All three of them have a possible complex interaction at cellular level. Smoking behavior makes it difficult to maintain infection control measures. Smoking is known to increase TB infection and also adversely affect treatment outcomes in TB. There is also upcoming evidence which suggests that smoking and TB increase the risk of severe Covid-19 symptoms. Simple infection control measures like, social distancing, cough etiquette, isolation, hand hygiene, quarantine, use of masks etc. play a pivotal role in prevention of these diseases. There is need of strengthening of the public health policies and incorporation of the Covid-19 safety awareness measures into the various national programmes.

Vaccines and variants: Modelling insights into emerging issues in COVID-19 epidemiology.

Mathematical modelling has played a pivotal role in understanding the epidemiology of and guiding public health responses to the ongoing coronavirus disease of 2019 (COVID-19) pandemic. Here, we review the role of epidemiological models in understanding evolving epidemic characteristics, including the effects of vaccination and Variants of Concern (VoC). We highlight ways in which models continue to provide important insights, including (1) calculating the herd immunity threshold and evaluating its limitations; (2) verifying that nascent vaccines can prevent severe disease, infection, and transmission but may be less efficacious against VoC; (3) determining optimal vaccine allocation strategies under efficacy and supply constraints; and (4) determining that VoC are more transmissible and lethal than previously circulating strains, and that immune escape may jeopardize vaccine-induced herd immunity. Finally, we explore how models can help us anticipate and prepare for future stages of COVID-19 epidemiology (and that of other diseases) through forecasts and scenario projections, given current uncertainties and data limitations.