Including Protease Inhibitors Research Articles

Pharmacological Strategies for Enzyme Inhibition in Disease Therapeutics: A Comprehensive Review

Abstract: Enzyme inhibition is a crucial pharmacological approach for treating various diseases as it targets enzymes involved in disease pathogenesis. This review explores the fundamental concepts of enzyme inhibition, including reversible and irreversible mechanisms, and the various types of enzymes, such as proteases, kinases, and polymerases, and their contributions to different disease states. The review discusses the design and production of enzyme inhibitors using methods like structurebased drug design, high-throughput screening, and rational drug design. The review also discusses the challenges and successes encountered in discovering and optimizing potent and selective enzyme inhibitors. Examples of enzyme inhibition's therapeutic benefits include protease inhibitors in HIV/AIDS therapy, kinase inhibitors in cancer treatment, and acetylcholinesterase inhibitors in Alzheimer's disease management. The review also examines possible side effects and limits of enzyme inhibition, focusing on ways to reduce off-target effects and make drugs more specific. At the end of the review, new trends and future possibilities in enzyme inhibition for treating diseases are talked about. These include personalized medicine, combination therapies, and new ways to get drugs into the body. By shedding light on the latest developments, challenges, and future directions, the review aims to contribute to the advancement of this vital field and revolutionize disease treatment modalities.

Investigating how HIV-1 antiretrovirals differentially behave as substrates and inhibitors of P-glycoprotein via molecular dynamics simulations

HIV-1 can rapidly infect the brain upon initial infection, establishing latent reservoirs that induce neuronal damage and/or death, resulting in HIV-Associated Neurocognitive Disorder. Though anti-HIV-1 antiretrovirals (ARVs) suppress viral load, the blood-brain barrier limits drug access to the brain, largely because of highly expressed efflux proteins like P-glycoprotein (P-gp). While no FDA-approved P-gp inhibitor currently exists, HIV-1 protease inhibitors show promise as partial P-gp inhibitors, potentially enhancing drug delivery to the brain. Herein, we employed docking and molecular dynamics simulations to elucidate key differences in P-gp’s interactions with several antiretrovirals, including protease inhibitors, with known inhibitory or substrate-like behaviors towards P-gp. Our results led us to hypothesize new mechanistic details of small-molecule efflux by and inhibition of P-gp, where the “Lower Pocket” in P-gp’s transmembrane domain serves as the primary initial site for small-molecule binding. Subsequently, this pocket merges with the more traditionally studied drug binding site—the “Upper Pocket”—thus funneling small-molecule drugs, such as ARVs, towards the Upper Pocket for efflux. Furthermore, our results reinforce the understanding that both binding energetics and changes in protein dynamics are crucial in discerning small molecules as non-substrates, substrates, or inhibitors of P-gp. Our findings indicate that interactions between P-gp and inhibitory ARVs induce bridging of transmembrane domain helices, impeding P-gp conformational changes and contributing to the inhibitory behavior of these ARVs. Overall, insights gained in this study could serve to guide the design of future P-gp-targeting therapeutics for a wide range of pathological conditions and diseases, including HIV-1.

Antiretroviral Treatment Gaps and Adherence Among People with HIV in the U.S. Medicare Program.

Approximately one-quarter of people with HIV (PWH) in the U.S. receive coverage through the Medicare program; however, no prior real-world study has examined antiretroviral therapy (ART) gaps and adherence and associated factors in this population. This retrospective cohort analysis used 2013-2018 national Medicare fee-for-service claims data to identify all PWH initiated on a new ART regimen including protease inhibitors [PI], non-nucleoside reverse transcriptase inhibitors [NNRTIs], or integrase strand transfer inhibitors [INSTIs] between 1/1/2014 and 12/31/2017. Study outcomes included ART adherence (based on proportion of days covered [PDC]), continuous treatment gaps ranging from 1 to 6 days to ≥ 180 days, and discontinuation (continuous gap ≥90 days) in the 12-month follow-up period. Multivariable regressions were used to assess factors associated with ART adherence and discontinuation. The final sample included 48,627 PWH (mean age: 54.5 years, 74.4% male, 47.5% White, 89.8% disabled). Approximately53.0% of PWH had a PDC ≥ 0.95, 30.2% had a PDC between 0.70 and < 0.95, and 16.8% had PDC < 0.70. Treatment gaps of at least ≥ 7-days (55.2%) and ≥ 30-days (26.2%) were common and 10.1% PWH discontinued treatment. Younger age, female sex, Black race, higher comorbidity score, mental health conditions, and substance use disorder were associated with higher odds of lower adherence and discontinuation (all p-values < 0.05). In conclusion, suboptimal adherence and treatment gaps in ART use were commonly observed among PWH in Medicare. Interventions and policies to mitigate barriers to adherence are urgently needed in this population to both improve their survival and increase the potential for ending the HIV epidemic in the US.

Host adaptation and specialization in Tetranychidae mites.

Composite generalist herbivores are comprised of host-adapted populations that retain the ability to shift hosts. The degree and overlap of mechanisms used by host-adapted generalist and specialist herbivores to overcome the same host plant defenses are largely unknown. Tetranychidae mites are exceptionally suited to address the relationship between host adaptation and specialization in herbivores as this group harbors closely related species with remarkably different host ranges-an extreme generalist the two-spotted spider mite (Tetranychus urticae Koch [Tu]) and the Solanaceous specialist Tetranychus evansi (Te). Here, we used tomato-adapted two-spotted spider mite (Tu-A) and Te populations to compare mechanisms underlying their host adaptation and specialization. We show that both mites attenuate induced tomato defenses, including protease inhibitors (PIs) that target mite cathepsin L digestive proteases. While Te solely relies on transcriptional attenuation of PI induction, Tu and Tu-A have elevated constitutive activity of cathepsin L proteases, making them less susceptible to plant anti-digestive proteins. Tu-A and Te also rely on detoxification of tomato constitutive defenses. Te uses esterase and P450 activities, while Tu-A depends on the activity of all major detoxification enzymatic classes to disarm tomato defensive compounds to a lesser extent. Thus, even though both Tu-A and Te use similar mechanisms to counteract tomato defenses, Te can better cope with them. This finding is congruent with the ecological and evolutionary times required to establish mite adaptation and specialization states, respectively.

CogiTx1: A novel subtilisin A inhibitor isolated from the sea anemone Condylactis gigantea belonging to the defensin 4 protein family

Subtilisin-like enzymes are recognized as key players in many infectious agents. In this context, its inhibitors are very valuable molecular lead compounds for structure based drug discovery and design. Marine invertebrates offer a great source of bioactive molecules, including protease inhibitors. In this work, we describe a new subtilisin inhibitor, from the sea anemone Condylactis gigantea (CogiTx1). CogiTx1 was purified using a combination of cation exchange chromatography, size exclusion chromatography and RP-HPLC chromatography. CogiTx1 it is a protein with 46 amino acid residues, with 4970.44 Da and three disulfide bridges. Is also able to inhibit subtilisin-like enzymes and pancreatic elastase. According to the amino acid sequence, it belongs to the defensin 4 family of proteins. The sequencing showed that CogiTx1 has an amidated C-terminal end, which was confirmed by the presence of the typical –XGR signal for amidation in the protein sequence deduced from the cDNA. This modification was described at protein level for the first time in this family of proteins. CogiTx1 is the first subtilisin inhibitor from the defensin 4 family and accordingly it has a folding consisting primarily in beta-strands in agreement with the analysis by CD and 3D modelling. Therefore, future in-depth functional studies may allow a more detailed characterization and will shed light on structure-function properties.

Aktivnost inhibitora tripsina u semenu sastrice (Lathyrus sativus L.)

Grass pea (Lathyrus sativus) is a valuable grain legume known for its high protein content and rich essential amino acid profile. Its exceptional characteristics such as drought tolerance, high adaptability to extreme conditions, disease resistance, and low cultivation inputs, make it particularly suitable for cultivation by resource-poor farmers. However, the potential use of grass pea is limited due to the presence of anti-nutritional factors, including protease inhibitors, especially trypsin inhibitor. This study aimed to develop a rapid and reliable method for measuring TI activity in seeds of grass pea and to investigate the influence of genotype and environment on trypsin inhibitor (TI) activity in seeds of grass pea. A set of 25 grass pea accessions from seven European countries was cultivated in Slovenia at the Agricultural Institute of Slovenia, and a set of 12 grass pea accessions from the Serbian gene bank was grown in Serbia at the Institute of Field and Vegetable Crops. The TI activity varied considerably among the grass pea accessions studied, with values ranging from 26.7 to 90.3 TUI/mg. To further evaluate the effects of environmental conditions on TI activity, eight grass pea accessions originating from Serbia were grown in both Slovenia and Serbia. The TI activity of the accessions grown in Slovenia ranged from 26.7 to 81.0 TUI/mg, while the activity of the accessions grown in Serbia ranged from 40.3 to 57.0 TUI/mg. The correlation of TI activity between grass pea accessions grown in Slovenia and those grown in Serbia was 0.39, with genotype diversity being the largest contributor (55.9%). This study provides a valuable insight into the variability of TI activity in grass pea and shows the possible influence of environmental conditions on this trait. However, since the data are only from a one-year field trial, further studies are needed to fully evaluate the influence of different environmental factors on TI activity.

Antiretroviral protease inhibitors induce features of cellular senescence that are reversible upon drug removal.

Antiretroviral drugs have dramatically improved the prognosis of HIV-infected patients, with strikingly reduced morbidity and mortality. However, long-term use can be associated with signs of premature aging. Highly active antiretroviral therapy generally comprises two nucleoside reverse transcriptase inhibitors (NRTIs), with one of three additional antiretroviral drug classes, including protease inhibitors (PIs). One commonality between mitochondrial dysfunction (induced by NRTIs) and defects in lamin A (induced by PIs) is they can cause or accelerate cellular senescence, a state of essentially irreversible growth arrest, and the secretion of many bioactive molecules collectively known as the senescence-associated secretory phenotype (SASP). We hypothesized that senescent cells increase following treatment with certain HIV therapies. We compared the effects of two distinct HIV PIs: ritonavir-boosted atazanavir (ATV/r) and ritonavir-boosted darunavir (DRN/r), used in combination treatments for HIV infection. Upon ATV/r, but not DRN/r, treatment, cells arrested growth, displayed multiple features of senescence, and expressed significantly upregulated levels of many SASP factors. Furthermore, mice receiving sustained ATV/r treatment showed an increase in senescent cells and age-related decline in physiological function. However, removing treatment reversed the features of senescence observed in vivo and cell culture. Given how these features disappeared with drug removal, certain features of senescence may not be prognostic as defined by an irreversible growth arrest. Importantly, for patients that are treated or have been treated with ATV/r, our data suggest that switching to another PI that does not promote premature aging conditions (DRN/r) may improve the associated age-related complications.

Identification of Novel Toxin Genes from the Stinging Nettle Caterpillar Parasa lepida (Cramer, 1799): Insights into the Evolution of Lepidoptera Toxins.

Simple SummaryMany caterpillar species can produce toxins that cause harmful reactions to humans, varying from mild irritation to death. Currently, there is very limited knowledge about caterpillar toxin diversity, because only a few species have been investigated. We used the transcriptome technique to identify candidate toxin genes from the nettle caterpillar Parasa lepida (Cramer, 1799). It is a common pest of oil palm, coconut, and mango in South and South-East Asia, which can cause severe pain and allergic responses to those in contact with them. We reported 168 candidate toxin genes. Most of them are members of the toxin genes families commonly recruited in animal venoms such as serine protease and serine protease inhibitors. However, we identified 21 novel genes encoding knottin-like peptides expressed at a high level in the transcriptome. Their predicted 3D structures are similar to neurotoxins in scorpion and tarantula. Our study suggests that P. lepida venom contains diverse toxin proteins that potentially cause allergic reactions and pain. This study sheds light on the hidden diversity of toxin proteins in caterpillar lineage, which could be future fruitful new drug sources.Many animal species can produce venom for defense, predation, and competition. The venom usually contains diverse peptide and protein toxins, including neurotoxins, proteolytic enzymes, protease inhibitors, and allergens. Some drugs for cancer, neurological disorders, and analgesics were developed based on animal toxin structures and functions. Several caterpillar species possess venoms that cause varying effects on humans both locally and systemically. However, toxins from only a few species have been investigated, limiting the full understanding of the Lepidoptera toxin diversity and evolution. We used the RNA-seq technique to identify toxin genes from the stinging nettle caterpillar, Parasa lepida (Cramer, 1799). We constructed a transcriptome from caterpillar urticating hairs and reported 34,968 unique transcripts. Using our toxin gene annotation pipeline, we identified 168 candidate toxin genes, including protease inhibitors, proteolytic enzymes, and allergens. The 21 P. lepida novel Knottin-like peptides, which do not show sequence similarity to any known peptide, have predicted 3D structures similar to tarantula, scorpion, and cone snail neurotoxins. We highlighted the importance of convergent evolution in the Lepidoptera toxin evolution and the possible mechanisms. This study opens a new path to understanding the hidden diversity of Lepidoptera toxins, which could be a fruitful source for developing new drugs.

Improvement of nutrient bioavailability in millets: Emphasis on the application of enzymes.

Millets are a traditional staple food of the dryland regions of the world and are rich in essential nutrients like protein, fatty acids, minerals, vitamins, and dietary fiber. Also, millets commonly synthesize a range of secondary metabolites to protect themselves against adverse conditions. These factors are collectively termed anti-nutritional factors and the existence of these factors in millets might reduce the accessibility of the nutrients in humans. Some of these factors include protease inhibitors, tannins, non-starch polysaccharides-glucans, phytates, and oxalates each of which might directly or indirectly affect the digestibility of nutrients. Methods like soaking, germination, autoclaving, debranning, and the addition of exogenous enzymes have been used to reduce the anti-nutritional factors and elevate the bioavailability of the nutrients. This review summarizes various methods that have been used to improve nutrient bioavailability, specifically emphasizing the use of enzymes to improve nutrient bioavailability from millets. © 2021 Society of Chemical Industry.

Modulated Electro-Hyperthermia Induces a Prominent Local Stress Response and Growth Inhibition in Mouse Breast Cancer Isografts.

Simple SummaryHere we investigated the most aggressive type of breast cancer (triple negative breast cancer (TNBC)) for which no effective therapies exist. Modulated electro-hyperthermia (mEHT) utilizes the altered bioelectric properties of tumors to implement a selective energy-transmission of an electromagnetic field and induce thermal and non-thermal anti-tumor effects. In our present study, repeated mEHT treatment effectively inhibited growth and proliferation and caused significant, destruction of TNBC tumors when applied alone without any other therapy in mice. Immunohistochemistry and multiplex analysis revealed that mEHT treatment induced protective mechanisms, like upregulation of heat shock proteins and other stress-related genes. Inhibition of these factors may serve as therapeutic approach to enhance the efficacy of mEHT. We were able to inhibit one of these protective proteins in cell culture. We aim to study the possibility of enhancing mEHT and other cancer therapies by inhibiting the identified protective stress response.Modulated electro-hyperthermia (mEHT) is a selective cancer treatment used in human oncology complementing other therapies. During mEHT, a focused electromagnetic field (EMF) is generated within the tumor inducing cell death by thermal and nonthermal effects. Here we investigated molecular changes elicited by mEHT using multiplex methods in an aggressive, therapy-resistant triple negative breast cancer (TNBC) model. 4T1/4T07 isografts inoculated orthotopically into female BALB/c mice were treated with mEHT three to five times. mEHT induced the upregulation of the stress-related Hsp70 and cleaved caspase-3 proteins, resulting in effective inhibition of tumor growth and proliferation. Several acute stress response proteins, including protease inhibitors, coagulation and heat shock factors, and complement family members, were among the most upregulated treatment-related genes/proteins as revealed by next-generation sequencing (NGS), Nanostring and mass spectrometry (MS). pathway analysis demonstrated that several of these proteins belong to the response to stimulus pathway. Cell culture treatments confirmed that the source of these proteins was the tumor cells. The heat-shock factor inhibitor KRIBB11 reduced mEHT-induced complement factor 4 (C4) mRNA increase. In conclusion, mEHT monotherapy induced tumor growth inhibition and a complex stress response. Inhibition of this stress response is likely to enhance the effectiveness of mEHT and other cancer treatments.

POS-694 RENAL TRANSOLANTATION STRUGGLES DURING COVID-19 ERA: DRUG/DRUG INTERACTION MAY LEAD TO GRAFT LOSS

Covid-19 treatment includes protease inhibitors (such as lopinavir/ritonavir) or other drugs that inhibit CYP3A and P-gp, with new concerns for drug interactions. CNIs and mTOR are cleared mainly via cytochrome P450 enzyme 3A5 (CYP3A5), CYP3A4, and P-glycoprotein (P-gp). CYP3A family is also involved in prednisolone metabolism. In HIV patients the micro dosing strategies for CNI prevented toxicity, dosage reduction of up to 120-fold and prolonged dosing intervals were needed in renal transplant recipients with HIV.

Antivirals including protease inhibitors linked with worse liver outcomes

Antivirals including protease inhibitors linked with worse liver outcomes

393. Prevalence of HIV in Patients Hospitalized for COVID-19 and Associated Mortality Outcomes: A Systematic Review and Meta-analysis

BackgroundAs of June 3rd, 2020, the number of confirmed cases of novel SARS-CoV-2, the causative agent of COVID-19, was approximately 6,538,456, with 386,503 deaths globally. Individuals with pre-existing conditions are particularly susceptible to and more likely to die from Covid-19. However, individuals with human immunodeficiency virus (HIV) are unique due to their use of antiretroviral therapy, including protease inhibitors, which have been used to treat COVID-19. We aimed to conduct a systematic review and meta-analysis exploring the prevalence and prevalence of HIV in patients hospitalized for COVID-19 and delineating the mortality rates.MethodsMEDLINE, SCOPUS, and Cochrane Library databases and medrxiv.org were searched from January 1st, 2020, to June 15th, 2020. Studies reporting on the prevalence of HIV among hospitalized COVID-19 patients among and outcome of mortality were extracted. Two reviewers independently extracted appropriate data of interest and assessed the risk of bias. All analyses were performed using random-effects models on log-transformed proportions and risk ratio estimates, and heterogeneity was quantified.ResultsA total of 144,795 hospitalized COVID-19 patients were identified from 14 studies (United States 8, Spain 3, China 1, Italy1, and Germany 1). The pooled prevalence of HIV in COVID-19 patients was 1.22 % [95% confidence interval (CI): 0.61%-2.43%)] translating to a 2-fold increase compared to the respective local-level pooled HIV prevalence in the general population of 0.65% (95% CI: 0.48%-0.89%. When we stratified the analysis by country, pooled HIV prevalence among COVID-19 patients in United States (1.43%, 95% CI: 0.98% -2.07%) was significantly higher compared to Spain (0.26%, 95% CI: 0.23%-0.29%) but not different from China (0.99 %, 95% CI: 0.25 %-3.85%). The pooled mortality rates in HIV-positive patients hospitalized for COVID-19 was 14.1 % 95% CI: 5.78%-30.50% and was substantially higher in the United States compared to other countries.ConclusionThe prevalence of HIV among COVID-19 patients may be higher compared to the general population, suggesting higher susceptibility to COVID-19. The mortality rates are high but vary significantly across countries.Disclosures All Authors: No reported disclosures

Molecular Structure of SARS-CoV-2 Virus and the Proposed Related Drugs

Background and Aim: Coronavirus Disease 2019 (COVID-19) is one of the most important viral diseases in the current decade which has caused widespread crisis worldwide. The disease was first reported on December 8, 2019 in Wuhan, Hubei Province, China, in the patients with acute respiratory distress syndrome. On January 8, 2020, the Centers for Disease Control and Prevention in China identified the virus, and it was named 2019-nCoV by the World Health Organization (WHO). The name of the disease was later changed to COVID-19. According to the International Committee on Taxonomy of Viruses, the virus is called SARS-CoV-2 which belongs to the family of Coronaviridae. The viruses of this family have caused outbreaks in several countries (SARS in 2003 and MERS in 2013) which have led to death and economic loss. Methods &amp; Materials: In this review study, COVID-19 was studied from various aspects including evolutionary process and molecular biology of its virus. The selected articles were examined based on the information available in the WHO database. SARS-CoV-2 proteins were identified molecularly and functionally using data analysis and bioinformatics methods, and then related drugs and their effects on virus replication and inhibition were investigated. Ethical Considerations: All ethical principles were observed in this study. Results: Studies on the structure of SARS-CoV-2 and drug therapies to inhibit the disease progression showed that the use of different pharmaceutical strategies is effective in treating COVID-19 depending on the progression of the disease. Molecular studies showed that the use of nucleoside analogues and protease inhibitors is effective in the course of the disease and intravenous immunoglobulin, aminoquinoline compounds, TMPRSS2 inhibitors and viral S protein can be effective in the early stages of disease. Conclusion: In order to control COVID-19, it is very important to study the structure of SARS-CoV-2 virus and its biology in the body. It is essential to identify the drugs that affect the virus based on its biological structure. Due to the structural changes of the virus and successive mutations in the virus genome as well as the emergence of resistant strains or highly contagious strains, further studies on the structure of SARS-CoV-2 and its changes in the body are recommended for designing pharmaceutical and therapeutic strategies. These strategies varies according to the stage of the disease, such that some drugs prevent the virus from entering the target cells in the early stages of the disease and other drugs, in combination with the virus’ surface glycoproteins, prevent the virus antigen from binding to receptors in the host cells. In the late stages of the disease, antiviral drugs including protease inhibitors and nucleoside analogues, interfere with the replication and structure of the virus. Due to intermittent changes in the virus and the development of drug-resistant viruses, it is important to continuously review virological and clinical studies and the performance of existing drugs against SARS-CoV-2.

Protease inhibitor-contaning antiretroviral therapy effects on QT interval and heart rate: a cross-sectional study

Abstract Background HIV has been associated with QT prolongation and it is believed this is from drug interaction as well as HIV infection. In vitro studies have showed that protease inhibitors block potassium channel HERG, suggesting that these drugs can cause drug-induced QT prolongation. Few case-reports have showed that protease inhibitors can cause drug-induced bradycardia, but data is still limited. Objective To describe the correlation between HIV infection and the duration of QT interval and to evaluate the effects that protease inhibitors-containing antiretroviral regime have on the duration of QT interval and cardiac frequency. Methods A cross-sectional study was made. Participants were assigned to 3 groups: HIV positive with antiretroviral therapy which included protease inhibitors (HIV+ with treatment), treatment-naïve HIV positive and HIV negative. None of the participants had past medical history of cardiovascular disease or use of known QT prolonging medication. 12-lead ECG were made to all participants. QT interval and cardiac frequency were measured to be compared between the 3 groups and the results were analyzed by the statistical test ANOVA. Results A total of 213 participants were enrolled, 84 in the HIV+ with treatment, 45 in the HIV+ treatment-naïve, and 84 HIV-. 63% of the participants were males. The treatment-naïve HIV+ group only had 5 female participants. This study did not find a relation between HIV infection or protease inhibitor use and an increase in the duration of QT interval in males. For females, this study found an association between HIV infection and longer QT interval duration, as seen in the mean of each group: 442.8 ms for treatment-naïve HIV+ group, 422.42 ms for protease inhibitor HIV+ with treatment group and 420.34 ms HIV- group. It was found that the use of antiretroviral regime with protease inhibitor was associated with a decrease in cardiac frequency, as seen in each group's cardiac frequencies: HIV+ with treatment group: 63.34 bpm, treatment-naïve HIV positive: 79.77 bpm and control group: 69.3 bpm. Conclusion We did not find correlation between HIV infection, the use of protease inhibitor-containing antiretroviral regime and QT interval prolongation in males, as opposed to females, where it was shown that QT interval duration is longer in the HIV+ treatment-naïve group. It was also observed that the use of protease inhibitor-containing antiretroviral regime was associated with a decrease in cardiac frequency. Funding Acknowledgement Type of funding source: None

Consequences of the COVID-19 pandemic on the continuum of care in a cohort of people living with HIV followed in a single center of Northern Italy

IntroductionDuring the COVID-19 pandemic, hospitals faced increasing pressure, where people living with HIV risked to either acquire SARS-CoV-2 and to interrupt the HIV continuum of care.MethodsThis is a retrospective, observational study. We compared the numbers of medical visits performed, antiretroviral drugs dispensed and the number of new HIV diagnosis and of hospitalizations in a cohort of people living with HIV (PLWH) followed by the Spedali Civili of Brescia between the bimester of the COVID-19 pandemic peak and the bimester of October–November 2019. Data were retrieved from administrative files and from paper and electronic clinical charts. Categorical variables were described using frequencies and percentages, while continuous variables were described using mean, median, and interquartile range (IQR) values. Means for continuous variables were compared using Student’s t-tests and the Mann–Whitney test. Proportions for categorical variables were compared using the χ2 test.ResultsAs of December 31st, 2019, a total of 3875 PLWH were followed in our clinic. Mean age was 51.4 ± 13 years old, where 28% were females and 18.8% non-Italian. Overall, 98.9% were on ART (n = 3834), 93% were viro-suppressed. A total of 1217 and 1162 patients had their visit scheduled at our out-patient HIV clinic during the two bimesters of 2019 and 2020, respectively. Comparing the two periods, we observed a raise of missed visits from 5 to 8% (p < 0.01), a reduction in the number of new HIV diagnosis from 6.4 in 2019 to 2.5 per month in 2020 (p = 0.01), a drop in ART dispensation and an increase of hospitalized HIV patients due to COVID-19. ART regimens including protease inhibitors (PIs) had a smaller average drop than ART not including PIs (16.6 vs 21.6%, p < 0.05). Whether this may be due to the perception of a possible efficacy of PIs on COVID19 is not known.ConclusionsOur experience highlights the importance of a resilient healthcare system and the need to implement new strategies in order to guarantee the continuum of HIV care even in the context of emergency.

Existence of SARS-CoV-2 Entry Molecules in the Oral Cavity.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor, angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and furin, which promote entry of the virus into the host cell, have been identified as determinants of SARS-CoV-2 infection. Dorsal tongue and gingiva, saliva, and tongue coating samples were examined to determine the presence of these molecules in the oral cavity. Immunohistochemical analyses showed that ACE2 was expressed in the stratified squamous epithelium of the dorsal tongue and gingiva. TMPRSS2 was strongly expressed in stratified squamous epithelium in the keratinized surface layer and detected in the saliva and tongue coating samples via Western blot. Furin was localized mainly in the lower layer of stratified squamous epithelium and detected in the saliva but not tongue coating. ACE2, TMPRSS2, and furin mRNA expression was observed in taste bud-derived cultured cells, which was similar to the immunofluorescence observations. These data showed that essential molecules for SARS-CoV-2 infection were abundant in the oral cavity. However, the database analysis showed that saliva also contains many protease inhibitors. Therefore, although the oral cavity may be the entry route for SARS-CoV-2, other factors including protease inhibitors in the saliva that inhibit viral entry should be considered.

Plant/Bacterial Virus-Based Drug Discovery, Drug Delivery, and Therapeutics.

Viruses have recently emerged as promising nanomaterials for biotechnological applications. One of the most important applications of viruses is phage display, which has already been employed to identify a broad range of potential therapeutic peptides and antibodies, as well as other biotechnologically relevant polypeptides (including protease inhibitors, minimizing proteins, and cell/organ targeting peptides). Additionally, their high stability, easily modifiable surface, and enormous diversity in shape and size, distinguish viruses from synthetic nanocarriers used for drug delivery. Indeed, several plant and bacterial viruses (e.g., phages) have been investigated and applied as drug carriers. The ability to remove the genetic material within the capsids of some plant viruses and phages produces empty viral-like particles that are replication-deficient and can be loaded with therapeutic agents. This review summarizes the current applications of plant viruses and phages in drug discovery and as drug delivery systems and includes a discussion of the present status of virus-based materials in clinical research, alongside the observed challenges and opportunities.

High Levels of HIV-1 Drug Resistance in Children Who Acquired HIV Infection Through Mother to Child Transmission in the Era of Option B+, Haiti, 2013 to 2014.

The main objective of this study was to determine the frequency and patterns of HIV drug resistance-associated mutations among children under 18 months of age born to HIV-1-positive mothers enrolled in the prevention of mother-to-child transmission services in Haiti. Between January 1, 2013 and December 31, 2014, HIV-positive remnant dried blood spots collected from children under 18 months of age for Early Infant Diagnosis at the National Public Health Laboratory were used for HIV-1 genotyping. HIV drug resistance mutations were analyzed using the Stanford Drug Resistance HIVdb program. Of the 3555 dried blood spots collected for Early Infant Diagnosis, 360 (10.1%) were HIV-positive and 355 were available for genotyping. Of these, 304 (85.6%) were successfully genotyped and 217 (71.4%) had ≥1 drug resistance mutation. Mutations conferring resistance to nucleoside reverse transcriptase inhibitor (NRTIs) and non-NRTIs were present in 40.5% (123) and 69.1% (210), respectively. The most frequent mutations were K103N/S (48.0%), M184V (37.5%), G190A/S (15.1%), and Y181C/G/V (14.1%). Predicted drug resistance analysis revealed that 68.8% of the children had high-level resistance to non-NRTIs and 11.5% had intermediate to high-level resistance to abacavir. This study showed high rates of resistance to NRTIs and non-NRTIs among newly HIV-diagnosed children in Haiti, suggesting that in the era of "Option B+" (initiation of lifelong combination antiretroviral therapy to pregnant women with HIV), the majority of children who acquire HIV infection through mother-to-child transmission of HIV have resistant HIV. These results have led the National HIV Program to revise the pediatric guidelines to include protease inhibitors in first-line regimens for all HIV-positive newborns.

In Vitro Susceptibility of Hepatitis C Virus Genotype 1 through 6 Clinical Isolates to the Pangenotypic NS3/4A Inhibitor Voxilaprevir.

Voxilaprevir is a direct-acting antiviral agent (DAA) that targets the NS3/4A protease of hepatitis C virus (HCV). High sequence diversity of HCV and inadequate drug exposure during unsuccessful treatment may lead to the accumulation of variants with reduced susceptibility to DAAs, including NS3/4A protease inhibitors such as voxilaprevir. The voxilaprevir susceptibility of clinical and laboratory strains of HCV was assessed. The NS3 protease regions of viruses belonging to 6 genotypes and 29 subtypes from 345 DAA-naive or -experienced (including protease inhibitor) patients and 344 genotype 1 to 6 replicons bearing engineered NS3 resistance-associated substitutions (RASs) were tested in transient-transfection assays. The median voxilaprevir 50% effective concentration against NS3 from protease inhibitor-naive patient samples ranged from 0.38 nM for genotype 1 to 5.8 nM for genotype 3. Voxilaprevir susceptibilities of HCV replicons with NS3 RASs were dependent on subtype background and the type and number of substitutions introduced. The majority of RASs known to confer resistance to other protease inhibitors had little to no impact on voxilaprevir susceptibility, except A156L, T, or V in genotype 1 to 4 which conferred >100-fold reductions but exhibited low replication capacity in most genotypes. These data support the use of voxilaprevir in combination with other DAAs in DAA-naive and DAA-experienced patients infected with any subtype of HCV.