Neutralizing antibody response after immunization with a COVID-19 bivalent vaccine: Insights to the future.

The raising of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants led to the use of COVID-19 bivalent vaccines, which include antigens of the wild-type (WT) virus, and of the Omicron strain. In this study, we aimed to evaluate the impact of bivalent vaccination on the neutralizing antibody (NAb) response. We enrolled 93 volunteers who had received three or four doses of monovalent vaccines based on the original virus (n = 61), or a booster shot with the bivalent vaccine (n = 32). Serum samples collected from volunteers were subjected to neutralization assays using the WT SARS-CoV-2, and Omicron subvariants. In addition, immunoinformatics to quantify and localize highly conserved NAb epitopes were performed. As main result, we observed that the neutralization titers of samples from individuals vaccinated with the bivalent vaccine were higher for the original virus, in comparison to their capacity of neutralizing the Omicron variant and its subvariants. NAb that recognize epitopes mostly conserved in the WT SARS-CoV-2 were boosted, while those that recognize epitopes mostly present in the Omicron variant, and subvariants were primed. These results indicate that formulation of future vaccines shall consider to target present viruses, and not viruses that no longer circulate.

Proposal of Model for Evaluation of Viral Kinetics of African/Asian/Brazilian-Zika virus Strains (Step Growth Curve) in Trophoblastic Cell Lines.

The Zika virus (ZIKV) epidemic brought new discoveries regarding arboviruses, especially flaviviruses, as ZIKV was described as sexually and vertically transmitted. The latter shows severe consequences for the embryo/fetus, such as congenital microcephaly and deficiency of the neural system, currently known as Congenital ZIKV Syndrome (CZS). To better understand ZIKV dynamics in trophoblastic cells present in the first trimester of pregnancy (BeWo, HTR-8, and control cell HuH-7), an experiment of viral kinetics was performed for African MR766 low passage and Asian-Brazilian IEC ZIKV lineages. The results were described independently and demonstrated that the three placental cells lines are permissive and susceptible to ZIKV. We noticed cytopathic effects that are typical in in vitro viral infection in BeWo and HTR-8. Regarding kinetics, MR766lp showed peaks of viral loads in 24 and 48 hpi for all cell types tested, as well as marked cells death after peak production. On the other hand, the HTR-8 lineage inoculated with ZIKV-IEC exhibited increased viral production in 144 hpi, with a peak between 24 and 96 hpi. Furthermore, IEC had peak variations of viral production for BeWo in 144 hpi. Considering such in vitro results, the hypothesis that maternal fetal transmission is probably a way of virus transmission between the mother and the embryo/fetus is maintained.

One-year surveillance of SARS-CoV-2 in wastewater from vulnerable urban communities in metropolitan São Paulo, Brazil.

The current COVID-19 pandemic has emphasized the vulnerability of communities living in the urban outskirts and informal settlements. The lack of reliable COVID-19 case data highlights the importance and application of wastewater-based epidemiology. This study aimed to monitor the COVID-19 trends in four vulnerable urban communities (slums and low-income neighborhoods) in metropolitan São Paulo by assessing the SARS-CoV-2 RNA viral load in wastewater. We analyzed 160 samples from May 2020 to June 2021 with weekly or fortnightly samplings. The samples were ultracentrifuged with glycine elution and quantified by N1/N2 SARS-CoV-2 RT-qPCR. The results of positivity were 100% (Paraisópolis, Heliópolis and Cidade Tiradentes) and 76.9% (Vila Brasilândia). The new case numbers of COVID-19, counted from the onset of symptoms, positively correlated with SARS-CoV-2 N1 viral loads from the two largest communities (p<0.001). SARS-CoV-2 infectivity was tested in Vero E6 cells after concentration with the two techniques, ultrafiltration (Centricon® Plus-70 10 kDa) and sucrose cushion ultracentrifugation, but none of the evaluated samples presented positive results. Next-generation sequencing (NGS) analysis from samples collected in March and August 2021 revealed the presence of the clade 20 J (lineage P.1) belonging to the most prevalent circulating variant in the country. Our results showed that wastewater surveillance data can be used as complementary indicators to monitor the dynamics and temporal trends of COVID-19. The infectivity test results strengthened the evidence of low risk of infection associated with SARS-CoV-2 in wastewater.

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.

Photoinduced Photosensitizer-Antibody Conjugates Kill HIV Env-Expressing Cells, Also Inactivating HIV.

HIV-infected cells persist for decades in patients administered with antiretroviral therapy (ART). Meanwhile, an alarming surge in drug-resistant HIV viruses has been occurring. Addressing these issues, we propose the application of photoimmunotherapy (PIT) against not only HIV Env-expressing cells but also HIV. Previously, we showed that a human anti-gp41 antibody (7B2) conjugated to cationic or anionic photosensitizers (PSs) could specifically target and kill the HIV Env-expressing cells. Here, our photolysis studies revealed that the binding of photoimmunoconjugates (PICs) on the membrane of HIV Env-expressing cells is sufficient to induce necrotic cell death due to physical damage to the membrane by singlet oxygen, which is independent of the type of PSs. This finding persuaded us to study the virus photoinactivation of PICs using two HIV-1 strains, X4 HIV-1 NL4-3 and JR-CSF virus. We observed that the PICs could destroy the viral strains, probably via physical damage on the HIV envelope. In conclusion, we report the application of PIT as a possible dual-tool for HIV immunotherapy and ART by killing HIV-expressing cells and cell-free HIV, respectively.

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.

Stochastic Modeling and Simulation of Viral Evolution.

RNA viruses comprise vast populations of closely related, but highly genetically diverse, entities known as quasispecies. Understanding the mechanisms by which this extreme diversity is generated and maintained is fundamental when approaching viral persistence and pathobiology in infected hosts. In this paper, we access quasispecies theory through a mathematical model based on the theory of multitype branching processes, to better understand the roles of mechanisms resulting in viral diversity, persistence and extinction. We accomplish this understanding by a combination of computational simulations and the theoretical analysis of the model. In order to perform the simulations, we have implemented the mathematical model into a computational platform capable of running simulations and presenting the results in a graphical format in real time. Among other things, we show that the establishment of virus populations may display four distinct regimes from its introduction into new hosts until achieving equilibrium or undergoing extinction. Also, we were able to simulate different fitness distributions representing distinct environments within a host which could either be favorable or hostile to the viral success. We addressed the most used mechanisms for explaining the extinction of RNA virus populations called lethal mutagenesis and mutational meltdown. We were able to demonstrate a correspondence between these two mechanisms implying the existence of a unifying principle leading to the extinction of RNA viruses.

Modulation of epigenetic factors during the early stages of HIV-1 infection in CD4+ T cells in vitro.

Several studies have related epigenetic mechanisms to HIV-1 latency. However, the epigenetic modifications of the host cell genome involved in the early stages of HIV-1 infection remain unclear. This study aimed to investigate epigenetic factors that are regulated at the beginning of HIV-1 infection in activated and resting CD4+ T cells. We analyzed the gene expression of 84 epigenetic targets, global DNA methylation, and HIV-1 replication kinetics for 36 h after infecting CD4+ T cells obtained from the blood of twelve healthy donors. The epigenetic targets aurora kinase B (AURKB), aurora kinase C (AURKC) and DNA methyltransferase 3B (DNMT3B), and the global DNA methylation profile are regulated during HIV-1 replication in CD4+ T cells, and this regulation can be influenced by the activation state of the cell at the time of infection. Approaches that affect the expression of these epigenetic targets could help current strategies to suppress HIV-1 replication.

Correction: Plasma Metabolomics Biosignature According to HIV Stage of Infection, Pace of Disease Progression, Viremia Level and Immunological Response to Treatment.

[This corrects the article DOI: 10.1371/journal.pone.0161920.].

Plasma Metabolomics Biosignature According to HIV Stage of Infection, Pace of Disease Progression, Viremia Level and Immunological Response to Treatment.

BACKGROUND: We evaluated plasma samples HIV-infected individuals with different phenotypic profile among five HIV-infected elite controllers and five rapid progressors after recent HIV infection and one year later and from 10 individuals subjected to antiretroviral therapy, five of whom were immunological non-responders (INR), before and after one year of antiretroviral treatment compared to 175 samples from HIV-negative patients. A targeted quantitative tandem mass spectrometry metabolomics approach was used in order to determine plasma metabolomics biosignature that may relate to HIV infection, pace of HIV disease progression, and immunological response to treatment. RESULTS: Twenty-five unique metabolites were identified, including five metabolites that could distinguish rapid progressors and INRs at baseline. Severe deregulation in acylcarnitine and sphingomyelin metabolism compatible with mitochondrial deficiencies was observed. ß-oxidation and sphingosine-1-phosphate-phosphatase-1 activity were down-regulated, whereas acyl-alkyl-containing phosphatidylcholines and alkylglyceronephosphate synthase levels were elevated in INRs. Evidence that elite controllers harbor an inborn error of metabolism (late-onset multiple acyl-coenzyme A dehydrogenase deficiency [MADD]) was detected. CONCLUSIONS: Blood-based markers from metabolomics show a very high accuracy of discriminating HIV infection between varieties of controls and have the ability to predict rapid disease progression or poor antiretroviral immunological response. These metabolites can be used as biomarkers of HIV natural evolution or treatment response and provide insight into the mechanisms of the disease.

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.

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

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™, and pol region 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.

Homogenous HIV-1 subtype B quasispecies in Brazilian men and women recently infected via heterosexual transmission.

HIV has extraordinary genetic mutability, both among individuals and at the population level. However, studies of primary HIV-1 infection and serum-converters indicate that the viral population is homogeneous at the sequence level, which suggests clonal HIV transmission. It remains unclear whether this feature applies to the female population. Ten single genome amplification sequences were generated from ten individuals (five females) with recent heterosexually acquired HIV infection as determined by the serologic testing algorithm for recent HIV seroconversion. Intra-individual genetic diversity was equally low in both genders (<2 %), with mean and median variations of 0.8 and 0 %, respectively. All of the subjects were infected with clade B. Three subjects (two females) appeared to be infected by two related viral populations, and four subjects harbored non-R5 strains. Our results support the hypothesis of clonal selection for sexual transmission of HIV-1 in both genders. Future studies that generate a larger number of clones, preferably by next generation deep sequencing, are needed to confirm these results.

Analysis of HIV-1 protease gene reveals frequent multiple infections followed by recombination among drug treated individuals living in São Paulo and Santos, Brazil.

The present study investigated the prevalence of HIV-1 multiple infections in a population composed by 47 patients under HAART failure and enrolled at the National DST/AIDS, Program, Ministry of Health, Brazil.Detection of multiple infections was done using a previously published RFLP assay for the HIV-1 protease gene, which is able of distinguishing between infections caused by a single or multiple HIV-1 subtypes. Samples with multiple infections were cloned, and sequence data submitted to phylogenetic analysis. We were able to identify 17 HIV-1 multiple infections out of 47 samples. Multiple infections were mostly composed by a mixture of recombinant viruses (94%), with only one case in which protease gene pure subtypes B and F were recovered. This is the first study that reports the prevalence of multiple infections and intersubtype recombinants in a population undergoing HAART in Brazil. Based on the data there was a steep increase of multiple infections after the introduction of the combined antiretroviral therapy in Brazil. Cases of multiple infections may be associated with HIV-1 genetic diversity through recombination allowing for the generation of viruses showing a combination of resistance mutations.