Associations between Varied Susceptibilities to PfATP4 Inhibitors and Genotypes in Ugandan Plasmodium falciparum Isolates.

Among novel compounds under recent investigation as potential new antimalarial drugs are three independently developed inhibitors of the Plasmodium falciparum P-type ATPase (PfATP4): KAE609 (cipargamin), PA92, and SJ733. We assessed ex vivo susceptibilities to these compounds of 374 fresh P. falciparum isolates collected in Tororo and Busia districts, Uganda, from 2016 to 2019. Median IC50s were 65 nM for SJ733, 9.1 nM for PA92, and 0.5 nM for KAE609. Sequencing of pfatp4 for 218 of these isolates demonstrated many nonsynonymous single nucleotide polymorphisms; the most frequent mutations were G1128R (69% of isolates mixed or mutant), Q1081K/R (68%), G223S (25%), N1045K (16%), and D1116G/N/Y (16%). The G223S mutation was associated with decreased susceptibility to SJ733, PA92, and KAE609. The D1116G/N/Y mutations were associated with decreased susceptibility to SJ733, and the presence of mutations at both codons 223 and 1116 was associated with decreased susceptibility to PA92 and SJ733. In all of these cases, absolute differences in susceptibilities of wild-type (WT) and mutant parasites were modest. Analysis of clones separated from mixed field isolates consistently identified mutant clones as less susceptible than WT. Analysis of isolates from other sites demonstrated the presence of the G223S and D1116G/N/Y mutations across Uganda. Our results indicate that malaria parasites circulating in Uganda have a number of polymorphisms in PfATP4 and that modestly decreased susceptibility to PfATP4 inhibitors is associated with some mutations now present in Ugandan parasites

Alirocumab, a Therapeutic Human Antibody to PCSK9, Does Not Affect CD81 Levels or Hepatitis C Virus Entry and Replication into Hepatocytes.

Proprotein convertase subtilisin/kexin type 9 (PSCK9) is secreted mainly from the liver and binds to the low-density lipoprotein receptor (LDLR), reducing LDLR availability and thus resulting in an increase in LDL-cholesterol. While the LDLR has been implicated in the cell entry process of the hepatitis C virus (HCV), overexpression of an artificial non-secreted, cell membrane-bound form of PCSK9 has also been shown to reduce surface expression of CD81, a major component of the HCV entry complex, leading to concerns that pharmacological inhibition of PCSK9 may increase susceptibility to HCV infection by increasing either CD81 or LDLR availability. Here, we evaluated effects of PCSK9 and PCSK9 blockade on CD81 levels and HCV entry with a physiologically relevant model using native secreted PCSK9 and a monoclonal antibody to PCSK9, alirocumab.Flow cytometry and Western blotting of human hepatocyte Huh-7 cells showed that, although LDLR levels were reduced when cells were exposed to increasing PCSK9 concentrations, there was no correlation between total or surface CD81 levels and the presence and amount of soluble PCSK9. Moreover, inhibiting PCSK9 with the monoclonal antibody alirocumab did not affect expression levels of CD81. In an in vitro model of HCV entry, addition of soluble PCSK9 or treatment with alirocumab had no effect on the ability of either lentiviral particles bearing the HCV glycoproteins or JFH-1 based cell culture virus to enter hepatocytes. Consistent with these in vitro findings, no differences were observed in hepatic CD81 levels using in vivo mouse models, including Pcsk9-/- mice compared with wild-type controls and hyperlipidemic mice homozygous for human Pcsk9 and heterozygous for Ldlr deletion, treated with either alirocumab or isotype control antibody.These results suggest that inhibition of PCSK9 with alirocumab has no effect on CD81 and does not result in increased susceptibility to HCV entry

Flow cytometry quantification of surface levels of LDLR (parts A and C) and CD81 (parts B and D) on Huh-7 cells, following incubation with the indicated concentrations of wild-type PCSK9 (parts A and B) or the gain-of-function PCSK9 D374Y mutant (parts C and D) and 300 nM alirocumab (monoclonal antibody to PCSK9; right panels) or isotype control monoclonal antibody (left panels) for 6 hours.

<p>LDLR, low-density lipoprotein receptor; PCSK9, proprotein convertase subtilisin/kexin type 9.</p

Effect of PCSK9 and alirocumab on the HCVcc full replication cycle (A) or HCV RNA replication cycle (B).

<p>Effect of PCSK9 and alirocumab on the HCVcc full replication cycle (A) or HCV RNA replication cycle (B).</p

Effect of soluble PCSK9, gain-of-function PCSK9 D374Y and alirocumab on HCVpp entry.

<p>Huh-7 cells were incubated for 6 hours with the indicated concentrations of wild-type PCSK9 or the gain-of-function PCSK9 D374Y mutant and alirocumab (monoclonal antibody to PCSK9) or isotype control monoclonal antibody (n = 3 replicates per treatment). Cells were then infected with HCVpp, incubated for 48–72 hours and intracellular luciferase activity was measured. Luciferase activity is shown as relative light units. Ab, antibody; HCVpp, hepatitis C virus pseudoparticles; PCSK9, proprotein convertase subtilisin/kexin type 9; RLU, relative light units; w/o, without.</p

Patients with post-baseline positive HCV test according to baseline status—data from alirocumab Phase 3 placebo- and ezetimibe-controlled trials.

<p>Patients with post-baseline positive HCV test according to baseline status—data from alirocumab Phase 3 placebo- and ezetimibe-controlled trials.</p

Effect of <i>Pcsk9</i> deletion on LDLR and CD81 levels in age- and sex-matched <i>Pcsk9</i>^<i>-/-</i> mice (n = 5) and their wild type littermates (n = 4).

<p>Shown are (A) mean ± SE serum LDL-C levels and (B) Western blot analysis of CD81 and LDLR levels in liver extracts with TfR as loading control, in animals sacrificed after a 4-hour fast. Lanes in panel B represent samples from individual mice. LDL-C, low-density lipoprotein cholesterol; LDLR, low-density lipoprotein receptor; PCSK9, proprotein convertase subtilisin/kexin type 9; SE, standard error; TfR, transferrin receptor; wt, wild type.</p

Western blot analysis on Huh-7 cells following incubation with decreasing concentrations of wild-type PCSK9 (500 nM, 50 nM and 5 nM) or gain-of-function PCSK9 D374Y mutant (20 nM, 2 nM and 0.2 nM) and 200 nM of alirocumab (monoclonal antibody to PCSK9) or isotype control monoclonal antibody for 6 hours.

<p>Lane 1 represents control cells incubated without PCSK9 protein or alirocumab/control Ab. Ab, antibody; LDLR, low-density lipoprotein receptor; PCSK9, proprotein convertase subtilisin/kexin type 9.</p

Nonstructural Protein 2 (nsP2) of Chikungunya Virus (CHIKV) Enhances Protective Immunity Mediated by a CHIKV Envelope Protein Expressing DNA Vaccine

Chikungunya virus (CHIKV) is an important emerging mosquito-borne alphavirus, indigenous to tropical Africa and Asia. It can cause epidemic fever and acute illness characterized by fever and arthralgias. The epidemic cycle of this infection is similar to dengue and urban yellow fever viral infections. The generation of an efficient vaccine against CHIKV is necessary to prevent and/or control the disease manifestations of the infection. In this report, we studied immune response against a CHIKV-envelope DNA vaccine (pEnv) and the role of the CHIKV nonstructural gene 2 (nsP2) as an adjuvant for the induction of protective immune responses in a relevant mouse challenge model. When injected with the CHIKV pEnv alone, 70% of the immunized mice survived CHIKV challenge, whereas when co-injected with pEnv+pnsP2, 90% of the mice survived viral challenge. Mice also exhibited a delayed onset signs of illness, and a marked decrease in morbidity, suggesting a nsP2 mediated adjuvant effect. Co-injection of the pnsP2 adjuvant with pEnv also qualitatively and quantitatively increased antigen specific neutralizing antibody responses compared to vaccination with pEnv alone. In sum, these novel data imply that the addition of nsP2 to the pEnv vaccine enhances anti-CHIKV-Env immune responses and maybe useful to include in future CHIKV clinical vaccination strategies