Andes hantavirus (ANDV) is an etiologic agent of hantavirus cardiopulmonary syndrome (HCPS), a severe disease characterized by fever, headache, and gastrointestinal symptoms that may progress to hypotension, pulmonary failure, and cardiac shock that results in a 25 to 40% case-fatality rate. Currently, there is no specific treatment or vaccine; however, several studies have shown that the generation of neutralizing antibody (Ab) responses strongly correlates with survival from HCPS in humans. In this study, we screened 27 ANDV convalescent HCPS patient sera for their capacity to bind and neutralize ANDV in vitro. One patient who showed high neutralizing titer was selected to isolate ANDV–glycoprotein (GP) Abs. ANDV-GP–specific memory B cells were single cell sorted, and recombinant immunoglobulin G antibodies were cloned and produced. Two monoclonal Abs (mAbs), JL16 and MIB22, potently recognized ANDV-GPs and neutralized ANDV. We examined the post-exposure efficacy of these two mAbs as a monotherapy or in combination therapy in a Syrian hamster model of ANDV-induced HCPS, and both mAbs protected 100% of animals from a lethal challenge dose. These data suggest that monotherapy with mAb JL16 or MIB22, or a cocktail of both, could be an effective post-exposure treatment for patients infected with ANDV-induced HCPS.

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The coronavirus disease 2019 (COVID19) pandemic has left researchers scrambling to identify the humoral immune correlates of protection from COVID-19. To date, the antibody mediated correlates of virus neutralization have been extensively studied. However, the extent that non-neutralizing functions contribute to anti-viral responses are ill defined. In this study, we profiled the anti-spike antibody subtype/subclass responses, along with neutralization and antibody-dependent natural killer cell functions in 83 blood samples collected between 4 and 201 days post-symptoms onset from a cohort of COVID-19 outpatients. We observed heterogeneous humoral responses against the acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Overall, anti-spike profiles were characterized by a rapid rise of IgA and sustained IgG titers. In addition, strong antibody-mediated natural killer effector responses correlated with milder disease and being female. While higher neutralization profiles were observed in males along with increased severity. These results give an insight into the underlying function of antibodies beyond neutralization and suggest that antibody-mediated natural killer cell activity is a key function of the humoral response against the SARS-CoV-2 spike protein.

BackgroundNew World Hantaviruses (NWHs) are the etiological agent underlying hantavirus cardiopulmonary syndrome (HCPS), a severe respiratory disease with high mortality rates in humans. In Panama, infections with Choclo Orthohantavirus (CHOV) cause a much milder illness characterized by higher seroprevalence and lower mortality rates. To date, the cytokine profiles and antibody responses associated with this milder form of HCPS have not been defined. Therefore, in this study, we examined immune serological profiles associated with CHOV infections.MethodsFor this retrospective study, sera from fifteen individuals with acute CHOV-induced HCPS, were analyzed alongside sera from fifteen convalescent phase individuals and thirty-three asymptomatic, CHOV-seropositive individuals. Cytokine profiles were analyzed by multiplex immunoassay. Antibody subclasses, binding, and neutralization against CHOV-glycoprotein (CHOV-GP) were evaluated by ELISA, and flow cytometry.ResultsHigh titers of IFNγ, IL-4, IL-8, and IL-10 serum cytokines were found in the acute individuals. Elevated IL-4 serum levels were found in convalescent and asymptomatic seropositive individuals. High titers of IgG1 subclass were observed across the three cohorts analyzed. Neutralizing antibody response against CHOV-GP was detectable in few acute individuals but was strong in both convalescent and asymptomatic seropositive individuals.ConclusionA Th1/Th2 cytokine signature is characteristic during acute mild HCPS caused by CHOV infection. High expression of Th2 and IL-8 cytokines are correlated with clinical parameters in acute mild HCPS. In addition, a strong IL-4 signature is associated with different cohorts, including asymptomatic individuals. Furthermore, asymptomatic individuals presented high titers of neutralizing antibodies.

Currently, the generation of cell lines for the production of recombinant proteins has the limitation of unstable gene expression due to the repeat-induced gene silencing or the loss of transgene copies resulting from recombination events. In this work, we developed a new strategy based on the sequential insertion of transgenes for generating stable clones producing high levels of a chimeric human follicle-stimulating hormone (hscFSH). Gene insertion was done by transducing HEK-293 cells with a lentiviral vector containing a bicistronic transcriptional unit for expressing hscFSH and GFP genes. Clone selection was performed by flow cytometry coupled to cell sorting, and the GFP gene was further removed by CRE-mediated site-specific recombination. High-producing clones of hscFSH were obtained after three rounds of lentiviral transduction. Expression levels increased in a step-wise manner from 7 to 23 pg/cell/day, with a relatively constant rate of 7 pg/cell/day in each round of transduction. The GFP gene was successfully removed from the cell genome without disturbing the hscFSH gene expression. Clones generated using this approach showed stable expression levels for more than two years. This is the first report describing the sequential insertion of transgenes as an alternative for increasing the expression levels of transformed cell lines. The methodology described here could notably impact on biotechnological industry by improving the capacity of mammalian cells to produce biopharmaceuticals.

in myocytes from basilar arteries; 4) myocyte BK channels in basilar arteries show the highest response to lithocholate; 5) lithocholate-induced dilation of basilar arteries is significantly higher than those measured in other intracranial territories. Collectively, our results demonstrate differential BK b1 expression, as well as its contribution to BK current phenotype and vasodilation by lithocholate, across the major branches of the Willis' circle. In particular, basilar arteries seem to be highly sensitive to vasoactive agents that act via BK b1 subunits. Support: HL104631 and AA11560 (AMD), and AHA Predoctoral Fellowship (GK).

Despite SARS-CoV-2 being a "novel" coronavirus, several studies suggest that detection of anti-spike IgG early in infection may be attributable to the amplification of humoral memory responses against seasonal hCoVs in severe COVID-19 patients. In this study, we examined this concept by characterizing anti-spike IgG from a cohort of non-hospitalized convalescent individuals with a spectrum of COVID-19 severity. We observed that anti-spike IgG levels positively correlated with disease severity, higher IgG cross-reactivity against betacoronaviruses (SARS-CoV-1 and OC43), and higher levels of proinflammatory Fc gamma receptor 2a and 3a (FcγR2a & FcγR3a) activation. In examining the levels of IgG targeting betacoronavirus conserved and immunodominant epitopes versus disease severity, we observed a positive correlation with the levels of IgG targeting the conserved S2'FP region, and an inverse correlation with two conserved epitopes around the heptad repeat (HR) 2 region. In comparing the levels of IgG targeting non-conserved epitopes, we observed that only one of three non-conserved immunodominant epitopes correlated with disease severity. Notably, the levels of IgG targeting the receptor binding domain (RBD) were inversely correlated with severity. Importantly, targeting of the RBD and HR2 regions have both been shown to mediate SARS-CoV-2 neutralization. These findings show that, aside from antibody (Ab) targeting of the RBD region, humoral memory responses against seasonal betacoronaviruses are potentially an important factor in dictating COVID-19 severity, with anti-HR2-dominant Ab profiles representing protective memory responses, while an anti-S2'FP dominant Ab profiles indicate deleterious recall responses. Though these profiles are masked in whole antigen profiling, these analyses suggest that distinct Ab memory responses are detectable with epitope targeting analysis. These findings have important implications for predicting severity of SARS-CoV-2 infections (primary and reinfections), and may predict vaccine efficacy in subpopulations with different dominant antibody epitope profiles.

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