Establishment of human post-vaccination SARS-CoV-2 standard reference sera.

There is a critical need to understand the effectiveness of serum elicited by different SARS-CoV-2 vaccines against SARS-CoV-2 variants. We describe the generation of reference reagents comprised of post-vaccination sera from recipients of different primary vaccines with or without different vaccine booster regimens in order to allow standardized characterization of SARS-CoV-2 neutralization in vitro. We prepared and pooled serum obtained from donors who received a either primary vaccine series alone, or a vaccination strategy that included primary and boosted immunization using available SARS-CoV-2 mRNA vaccines (BNT162b2, Pfizer and mRNA-1273, Moderna), replication-incompetent adenovirus type 26 vaccine (Ad26.COV2·S, Johnson and Johnson), or recombinant baculovirus-expressed spike protein in a nanoparticle vaccine plus Matrix-M adjuvant (NVX-CoV2373, Novavax). No subjects had a history of clinical SARS-CoV-2 infection, and sera were screened with confirmation that there were no nucleocapsid antibodies detected to suggest natural infection. Twice frozen sera were aliquoted, and serum antibodies were characterized for SARS-CoV-2 spike protein binding (estimated WHO antibody binding units/ml), spike protein competition for ACE-2 binding, and SARS-CoV-2 spike protein pseudotyped lentivirus transduction. These reagents are available for distribution to the research community (BEI Resources), and should allow the direct comparison of antibody neutralization results between different laboratories. Further, these sera are an important tool to evaluate the functional neutralization activity of vaccine-induced antibodies against emerging SARS-CoV-2 variants of concern. IMPORTANCE: The explosion of COVID-19 demonstrated how novel coronaviruses can rapidly spread and evolve following introduction into human hosts. The extent of vaccine- and infection-induced protection against infection and disease severity is reduced over time due to the fall in concentration, and due to emerging variants that have altered antibody binding regions on the viral envelope spike protein. Here, we pooled sera obtained from individuals who were immunized with different SARS-CoV-2 vaccines and who did not have clinical or serologic evidence of prior infection. The sera pools were characterized for direct spike protein binding, blockade of virus-receptor binding, and neutralization of spike protein pseudotyped lentiviruses. These sera pools were aliquoted and are available to allow inter-laboratory comparison of results and to provide a tool to determine the effectiveness of prior vaccines in recognizing and neutralizing emerging variants of concern.

A platform of assays for the discovery of anti-Zika small-molecules with activity in a 3D-bioprinted outer-blood-retina model.

The global health emergency posed by the outbreak of Zika virus (ZIKV), an arthropod-borne flavivirus causing severe neonatal neurological conditions, has subsided, but there continues to be transmission of ZIKV in endemic regions. As such, there is still a medical need for discovering and developing therapeutical interventions against ZIKV. To identify small-molecule compounds that inhibit ZIKV disease and transmission, we screened multiple small-molecule collections, mostly derived from natural products, for their ability to inhibit wild-type ZIKV. As a primary high-throughput screen, we used a viral cytopathic effect (CPE) inhibition assay conducted in Vero cells that was optimized and miniaturized to a 1536-well format. Suitably active compounds identified from the primary screen were tested in a panel of orthogonal assays using recombinant Zika viruses, including a ZIKV Renilla luciferase reporter assay and a ZIKV mCherry reporter system. Compounds that were active in the wild-type ZIKV inhibition and ZIKV reporter assays were further evaluated for their inhibitory effects against other flaviviruses. Lastly, we demonstrated that wild-type ZIKV is able to infect a 3D-bioprinted outer-blood-retina barrier tissue model and disrupt its barrier function, as measured by electrical resistance. One of the identified compounds (3-Acetyl-13-deoxyphomenone, NCGC00380955) was able to prevent the pathological effects of the viral infection on this clinically relevant ZIKV infection model.

Deep learning identifies synergistic drug combinations for treating COVID-19.

Effective treatments for COVID-19 are urgently needed. However, discovering single-agent therapies with activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been challenging. Combination therapies play an important role in antiviral therapies, due to their improved efficacy and reduced toxicity. Recent approaches have applied deep learning to identify synergistic drug combinations for diseases with vast preexisting datasets, but these are not applicable to new diseases with limited combination data, such as COVID-19. Given that drug synergy often occurs through inhibition of discrete biological targets, here we propose a neural network architecture that jointly learns drug-target interaction and drug-drug synergy. The model consists of two parts: a drug-target interaction module and a target-disease association module. This design enables the model to utilize drug-target interaction data and single-agent antiviral activity data, in addition to available drug-drug combination datasets, which may be small in nature. By incorporating additional biological information, our model performs significantly better in synergy prediction accuracy than previous methods with limited drug combination training data. We empirically validated our model predictions and discovered two drug combinations, remdesivir and reserpine as well as remdesivir and IQ-1S, which display strong antiviral SARS-CoV-2 synergy in vitro. Our approach, which was applied here to address the urgent threat of COVID-19, can be readily extended to other diseases for which a dearth of chemical-chemical combination data exists.

Chemoprotective antimalarials identified through quantitative high-throughput screening of Plasmodium blood and liver stage parasites.

The spread of Plasmodium falciparum parasites resistant to most first-line antimalarials creates an imperative to enrich the drug discovery pipeline, preferably with curative compounds that can also act prophylactically. We report a phenotypic quantitative high-throughput screen (qHTS), based on concentration-response curves, which was designed to identify compounds active against Plasmodium liver and asexual blood stage parasites. Our qHTS screened over 450,000 compounds, tested across a range of 5 to 11 concentrations, for activity against Plasmodium falciparum asexual blood stages. Active compounds were then filtered for unique structures and drug-like properties and subsequently screened in a P. berghei liver stage assay to identify novel dual-active antiplasmodial chemotypes. Hits from thiadiazine and pyrimidine azepine chemotypes were subsequently prioritized for resistance selection studies, yielding distinct mutations in P. falciparum cytochrome b, a validated antimalarial drug target. The thiadiazine chemotype was subjected to an initial medicinal chemistry campaign, yielding a metabolically stable analog with sub-micromolar potency. Our qHTS methodology and resulting dataset provides a large-scale resource to investigate Plasmodium liver and asexual blood stage parasite biology and inform further research to develop novel chemotypes as causal prophylactic antimalarials.

Synergistic and Antagonistic Drug Combinations against SARS-CoV-2.

Antiviral drug development for coronavirus disease 2019 (COVID-19) is occurring at an unprecedented pace, yet there are still limited therapeutic options for treating this disease. We hypothesized that combining drugs with independent mechanisms of action could result in synergy against SARS-CoV-2, thus generating better antiviral efficacy. Using in silico approaches, we prioritized 73 combinations of 32 drugs with potential activity against SARS-CoV-2 and then tested them in vitro. Sixteen synergistic and eight antagonistic combinations were identified; among 16 synergistic cases, combinations of the US Food and Drug Administration (FDA)-approved drug nitazoxanide with remdesivir, amodiaquine, or umifenovir were most notable, all exhibiting significant synergy against SARS-CoV-2 in a cell model. However, the combination of remdesivir and lysosomotropic drugs, such as hydroxychloroquine, demonstrated strong antagonism. Overall, these results highlight the utility of drug repurposing and preclinical testing of drug combinations for discovering potential therapies to treat COVID-19.

Sulfamethoxazole Levels in HIV-Exposed Uninfected Ugandan Children.

Trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis in HIV-uninfected, exposed (HUE) children variably reduces clinical malaria burden despite antifolate resistance, but data regarding achieved serum levels and adherence are lacking. Serum samples from 70 HUE children aged 3-12 months from Rakai, Uganda, enrolled in an observational study were assayed for random SMX levels using a colorimetric assay. Adherence with TMP-SMX prophylaxis data (yes/no) was also collected. Of 148 visits with concurrent SMX levels available, 56% had self-reported adherence with TMP-SMX therapy. Among these 82 visits, mean (standard deviation) level was 19.78 (19.22) µg/mL, but 33% had SMX levels below half maximal inhibitory concentrations (IC50) for Plasmodium falciparum with some, but not all, of the reported antifolate resistance mutations reported in Uganda. With TMP-SMX prophylaxis, suboptimal adherence is concerning. Sulfamethoxazole levels below IC50s required to overcome malaria parasites with multiple antifolate resistance mutations may be significant. Further study of TMP-SMX in this context is needed.

Mechanism of splenic cell death and host mortality in a Plasmodium yoelii malaria model.

Malaria is a fatal disease that displays a spectrum of symptoms and severity, which are determined by complex host-parasite interactions. It has been difficult to study the effects of parasite strains on disease severity in human infections, but the mechanisms leading to specific disease phenotypes can be investigated using strains of rodent malaria parasites that cause different disease symptoms in inbred mice. Using a unique mouse malaria model, here we investigated the mechanisms of splenic cell death and their relationship to control of parasitemia and host mortality. C57BL/6 mice infected with Plasmodium yoelii nigeriensis N67C display high levels of pro-inflammatory cytokines and chemokines (IL-6, IFN-γ, TNF-α, CXCL1, and CCL2) and extensive splenic damage with dramatic reduction of splenic cell populations. These disease phenotypes were rescued in RAG2-/-, IFN-γ-/-, or T cell depleted mice, suggesting IFN-γ and T cell mediated disease mechanisms. Additionally, apoptosis was one of the major pathways involved in splenic cell death, which coincides with the peaks of pro-inflammatory cytokines. Our results demonstrate the critical roles of T cells and IFN-γ in mediating splenic cell apoptosis, parasitemia control, and host lethality and thus may provide important insights for preventing/reducing morbidity associated with severe malaria in humans.

C-terminal proteolysis of prenylated proteins in trypanosomatids and RNA interference of enzymes required for the post-translational processing pathway of farnesylated proteins.

The C-terminal "CaaX"-motif-containing proteins usually undergo three sequential post-translational processing steps: (1) attachment of a prenyl group to the cysteine residue; (2) proteolytic removal of the last three amino acids "aaX"; (3) methyl esterification of the exposed alpha-carboxyl group of the prenyl-cysteine residue. The Trypanosoma brucei and Leishmania major Ras converting enzyme 1 (RCE1) orthologs of 302 and 285 amino acids-proteins, respectively, have only 13-20% sequence identity to those from other species but contain the critical residues for the activity found in other orthologs. The Trypanosoma brucei a-factor converting enzyme 1 (AFC1) ortholog consists of 427 amino acids with 29-33% sequence identity to those of other species and contains the consensus HExxH zinc-binding motif. The trypanosomatid RCE1 and AFC1 orthologs contain predicted transmembrane regions like other species. Membranes from Sf9 cells expressing the RCE1 ortholog of T. brucei or L. major showed proteolytic activity against farnesylated RAS-CVIM, whereas membranes containing T. brucei AFC1 ortholog were inactive. The results suggest that RCE1 is responsible for proteolytic removal of the C-terminal aaX from prenyl-CaaX proteins in these parasites. All the three enzymatic post-translational processes are thought to be required for proper cellular functioning of CaaX-proteins in eukaryotic cells. We carried out RNA interference experiments in Trypanosoma brucei of the enzymes involved in farnesyl protein post-translational modification to evaluate their importance in cell proliferation. Knockdown of T. brucei PFT beta subunit and RCE1 mRNAs resulted in >20-fold suppression of cell growth and dramatic morphologic changes. Knockdown of PPMT mRNA caused less dramatic effects on growth but induced noticeable changes in cell morphology.

Thematic review series: lipid posttranslational modifications. Fighting parasitic disease by blocking protein farnesylation.

Protein farnesylation is a form of posttranslational modification that occurs in most, if not all, eukaryotic cells. Inhibitors of protein farnesyltransferase (PFTIs) have been developed as anticancer chemotherapeutic agents. Using the knowledge gained from the development of PFTIs for the treatment of cancer, researchers are currently investigating the use of PFTIs for the treatment of eukaryotic pathogens. This "piggy-back" approach not only accelerates the development of a chemotherapeutic agent for protozoan pathogens but is also a means of mitigating the costs associated with de novo drug design. PFTIs have already been shown to be efficacious in the treatment of eukaryotic pathogens in animal models, including both Trypanosoma brucei, the causative agent of African sleeping sickness, and Plasmodium falciparum, one of the causative agents of malaria. Here, current evidence and progress are summarized that support the targeting of protein farnesyltransferase for the treatment of parasitic diseases.

Leishmania inactivation in human pheresis platelets by a psoralen (amotosalen HCl) and long-wavelength ultraviolet irradiation.

BACKGROUND: Leishmania spp. are protozoans that cause skin and visceral diseases. Leishmania are obligate intracellular parasites of mononuclear phagocytes and have been documented to be transmitted by blood transfusion. STUDY DESIGN AND METHODS: This study examines whether Leishmania can be inactivated in human platelet (PLT) concentrates by a photochemical treatment process that is applicable to blood bank use. Human PLT concentrates were contaminated with Leishmania mexicana metacyclic promastigotes or mouse-derived Leishmania major amastigotes and were exposed to long-wavelength ultraviolet (UV) A light (320-400 nm) plus the psoralen amotosalen HCl. RESULTS: Neither treatment with amotosalen nor UVA alone had an effect on Leishmania viability; however, treatment with 150 micromol per L amotosalen plus 3 J per cm(2) UVA inactivated both metacyclic promastigotes and amastigotes to undetectable levels, more than a 10,000-fold reduction in viability. CONCLUSIONS: This study demonstrates the effectiveness of photochemical treatment to inactivate Leishmania in PLT concentrates intended for transfusion. Both metacylic promastigotes, which represent the infectious form from the sand fly vector, and amastigotes, which represent the form that grows in mononuclear phagocytes, were extremely susceptible to photochemical inactivation by this process. Thus, the photochemical treatment of PLT concentrates inactivates both forms of Leishmania that would be expected to circulate in blood products collected from infected donors.

Protein farnesyl transferase inhibitors for the treatment of malaria and African trypanosomiasis.

Protein farnesyl transferase inhibitors (PFTIs) have been developed as oncology therapeutics but recent studies have supported the use of PFTIs for the treatment of eukaryotic pathogens. Data supporting PFTIs for the treatment of African sleeping sickness caused by Trypanosoma brucei sp, and for the therapy of malaria caused by Plasmodium spp is reviewed. Protein prenylation in T. brucei and P. falciparum has been studied using a variety of techniques, including recombinant and native enzyme assays. Studies have demonstrated farnesylation and geranylgeranylation in these parasites. A variety of PFTIs have shown growth inhibition activity and killing of T. brucei and P. falciparum, yet not all mammalian PFTIs are active on parasitic PFTs. Protein farnesyl transferase as well as protein geranylgeranyl transferase type II enzymatic activities have been demonstrated in T brucei and P. falciparum, but protein geranylgeranyl transferase type I activity may be lacking from these parasites, perhaps explaining the extreme sensitivity of these organisms to PFTIs compared with mammalian cells. Given that PFTIs are relatively non-toxic in short-term administration to humans, PFTIs specific to parasites are not required for therapy. Thus, the challenge in PFTI drug development is not to identify selective antiparasite compounds, but to identify compounds with sufficient potency and pharmacokinetic properties to produce satisfactory drugs for malaria and African sleeping sickness.

Resistance to a protein farnesyltransferase inhibitor in Plasmodium falciparum.

The post-translational farnesylation of proteins serves to anchor a subset of intracellular proteins to membranes in eukaryotic organisms and also promotes protein-protein interactions. Inhibition of protein farnesyltransferase (PFT) is lethal to the pathogenic protozoa Plasmodium falciparum. Parasites were isolated that were resistant to BMS-388891, a tetrahydroquinoline (THQ) PFT inhibitor. Resistance was associated with a 12-fold decrease in drug susceptibility. Genotypic analysis revealed a single point mutation in the beta subunit in resistant parasites. The resultant tyrosine 837 to cysteine alteration in the beta subunit corresponded to the binding site for the THQ and peptide substrate. Biochemical analysis of Y837C-PFT demonstrated a 13-fold increase in BMS-388891 concentration necessary for inhibiting 50% of the enzyme activity. These data are consistent with PFT as the target of BMS-388891 in P. falciparum and suggest that PFT inhibitors should be combined with other antimalarial agents for effective therapy.