SARS-CoV-2 infection and replication kinetics in different human cell types: The role of autophagy, cellular metabolism and ACE2 expression.

AIMS: This study evaluated SARS-CoV-2 replication in human cell lines derived from various tissues and investigated molecular mechanisms related to viral infection susceptibility and replication. MAIN METHODS: SARS-CoV-2 replication in BEAS-2B and A549 (respiratory tract), HEK-293 T (kidney), HuH7 (liver), SH-SY5Y (brain), MCF7 (breast), Huvec (endothelial) and Caco-2 (intestine) was evaluated by RT-qPCR. Concomitantly, expression levels of ACE2 (Angiotensin Converting Enzyme) and TMPRSS2 were assessed through RT-qPCR and western blot. Proteins related to autophagy and mitochondrial metabolism were monitored in uninfected cells to characterize the cellular metabolism of each cell line. The effect of ACE2 overexpression on viral replication in pulmonary cells was also investigated. KEY FINDINGS: Our data show that HuH7, Caco-2 and MCF7 presented a higher viral load compared to the other cell lines. The increased susceptibility to SARS-CoV-2 infection seems to be associated not only with the differential levels of proteins intrinsically related to energetic metabolism, such as ATP synthase, citrate synthase, COX and NDUFS2 but also with the considerably higher TMPRSS2 mRNA expression. The two least susceptible cell types, BEAS-2B and A549, showed drastically increased SARS-CoV-2 replication capacity when ACE2 was overexpressed. These modified cell lines are relevant for studying SARS-CoV-2 replication in vitro. SIGNIFICANCE: Our data not only reinforce that TMPRSS2 expression and cellular energy metabolism are important molecular mechanisms for SARS-CoV-2 infection and replication, but also indicate that HuH7, MCF7 and Caco-2 are suitable models for mechanistic studies of COVID-19. Moreover, pulmonary cells overexpressing ACE2 can be used to understand mechanisms associated with SARS-CoV-2 replication.

17ß-estradiol reduces SARS-CoV-2 infection in vitro.

The COVID-19 has originated from Wuhan, China, in December 2019 and has been affecting the public health system, society, and economy in an unheard-of manner. There is no specific treatment or vaccine available for COVID-19. Previous data showed that men are more affected than women by COVID-19, then we hypothesized whether sex hormones could be protecting the female organism against the infection. VERO E6 cells have been commonly used as in vitro model for SARS-CoV-2 infection. In our experimental approach, we have treated VERO E6 cells with 17ß-estradiol to evaluate the modulation of SARS-CoV-2 infection in this cell line. Here we demonstrated that estrogen protein receptors ERα, ERß, and GPER1 are expressed by VERO E6 cells and could be used to study the effects of this steroid hormone. Previous and 24-hours post-infection, cells treated with 17ß-estradiol revealed a reduction in the viral load. Afterward, we found that SARS-CoV-2 infection per se results in ACE2 and TMPRSS2 increased gene expression in VERO E6-cell, which could be generating a cycle of virus infection in host cells. The estrogen treatment reduces the levels of the TMPRSS2, which are involved with SARS-CoV-2 infectiveness capacity, and hence, reducing the pathogenicity/genesis. These data suggest that estrogen could be a potential therapeutic target promoting cell protection against SARS-CoV-2. This opens new possibilities for further studies on 17ß-estradiol in human cell lines infected by SARS-CoV-2 and at least in part, explain why men developed a more severe COVID-19 compared to women.

Photoimmunotherapy Using Cationic and Anionic Photosensitizer-Antibody Conjugates against HIV Env-Expressing Cells.

Different therapeutic strategies have been investigated to target and eliminate HIV-1-infected cells by using armed antibodies specific to viral proteins, with varying degrees of success. Herein, we propose a new strategy by combining photodynamic therapy (PDT) with HIV Env-targeted immunotherapy, and refer to it as HIV photoimmunotherapy (PIT). A human anti-gp41 antibody (7B2) was conjugated to two photosensitizers (PSs) with different charges through different linking strategies; "Click" conjugation by using an azide-bearing porphyrin attached via a disulfide bridge linker with a drug-to-antibody ratio (DAR) of exactly 4, and "Lysine" conjugation by using phthalocyanine IRDye 700DX dye with average DARs of 2.1, 3.0 and 4.4. These photo-immunoconjugates (PICs) were compared via biochemical and immunological characterizations regarding the dosimetry, solubility, and cell targeting. Photo-induced cytotoxicity of the PICs were compared using assays for apoptosis, reactive oxygen species (ROS), photo-cytotoxicity, and confocal microscopy. Targeted phototoxicity seems to be primarily dependent on the binding of PS-antibody to the HIV antigen on the cell membrane, whilst being independent of the PS type. This is the first report of the application of PIT for HIV immunotherapy by killing HIV Env-expressing cells.

HIV-1 infection modulates IL-24 expression which contributes to cell apoptosis in vitro.

Although interleukin-24 (IL-24) has been extensively explored in the immunopathologies of autoimmune diseases, neoplasms, and infections, its role in HIV-1 infection has not been thoroughly elucidated to date. Therefore, the objective of this study was to evaluate the gene and protein expressions of IL-24 at the initial moments of HIV infection in PBMCs. Due to the pro-apoptotic role of IL-24, we evaluated the protein expression of caspase-3, as well as Annexin V/Propidium Iodide flow cytometry and phosphorylation of ERK, which may induce an apoptotic signal block when phosphorylated. The results of this study demonstrated that HIV-1 infection had an impact on the gene and protein expressions of IL-24 and ERK. Annexin V/Propidium Iodide assay demonstrated decrease in the mechanisms of apoptosis in infected cells after incubation of IL-24 neutralizing antibody. Studies on how HIV-1 regulates IL-24 expression may play a role in characterizing viral persistence mechanisms and designing antiretroviral strategies.

High prevalence and incidence of HIV-1 in a counseling and testing center in the city of Itajaí, Brazil

ABSTRACT Itajaí is a port city in southern Brazil with one of the highest incidence and mortality rates from AIDS in the country. The prevalence and incidence of HIV infection were investigated in 1085 of 3196 new HIV-1 infection cases evaluated in the counseling and testing center of Itajaí from January 2002 to August 2008. Recent infections were assessed using the BED(tm), and polregion sequencing was performed in 76 samples. The prevalence ranged from 3.08% to 6.17% among women and from 10.26% to 17.36% among men. A total of 17% of infections were classified as recent, with annual incidence varying from 1.6% to 4.8 per 100 patient/year among women and from 2.05% to 8.5 per 100 patient/year among men. Pol sequences were obtained from 38 randomly recent infections selected individuals: 71% were infected by subtype C, 24% B, 2% D, and 2% F1. Among 38 subjects with established infection, 76% were subtype C, and 24% B. Transmitted drug resistance was detected in 18.4% of recent infection subjects (7.8% to nucleoside analog reverse-transcriptase inhibitors, 5.2% to non-nucleoside reverse-transcriptase inhibitors, and 5.2% protease inhibitors) and 5.2% of subjects with established infection had nucleoside analog reverse-transcriptase inhibitors resistance. The high prevalence and incidence of HIV infection in this region is unprecedented in studies involving cases evaluated in the counseling and testing centers in Brazil.