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In this perspective article, we describe the current status of lipid tools for studying host lipid-virus interactions at the cellular level. We discuss the potential lipidomic changes that viral infections impose on host cells and then outline the tools available and the resulting options to investigate the host cell lipid interactome. The future outcome will reveal new targets for treating virus infections.

Immunization in pregnancy is a critical tool that can be leveraged to protect the infant with an immature immune system but how vaccine-induced antibodies transfer to the placenta and protect the maternal-fetal dyad remains unclear. Here, we compare matched maternal-infant cord blood from individuals who in pregnancy received mRNA COVID-19 vaccine, were infected by SARS-CoV-2, or had the combination of these two immune exposures. We find that some but not all antibody neutralizing activities and Fc effector functions are enriched with vaccination compared to infection. Preferential transport to the fetus of Fc functions and not neutralization is observed. Immunization compared to infection enriches IgG1-mediated antibody functions with changes in antibody post-translational sialylation and fucosylation that impact fetal more than maternal antibody functional potency. Thus, vaccine enhanced antibody functional magnitude, potency and breadth in the fetus are driven more by antibody glycosylation and Fc effector functions compared to maternal responses, highlighting prenatal opportunities to safeguard newborns as SARS-CoV-2 becomes endemic.One Sentence SummarySARS-CoV-2 vaccination in pregnancy induces diverging maternal and infant cord antibody functions

The unprecedented severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has called for substantial investigations into the capacity of the human immune system to protect against reinfection and keep pace with the evolution of SARS-CoV-2. We evaluated the magnitude and durability of the SARS-CoV-2–specific antibody responses against parental WA-1 SARS-CoV-2 receptor-binding domain (RBD) and a representative variant of concern (VoC) RBD using antibodies from 2 antibody compartments: long-lived plasma cell–derived plasma antibodies and antibodies encoded by SARS-CoV-2–specific memory B cells (MBCs). Thirty-five participants naturally infected with SARS-CoV-2 were evaluated; although only 25 of 35 participants had VoC RBD–reactive plasma antibodies, 34 of 35 (97%) participants had VoC RBD–reactive MBC-derived antibodies. Our finding that 97% of previously infected individuals have MBCs specific for variant RBDs provides reason for optimism regarding the capacity of vaccination, prior infection, and/or both, to elicit immunity with the capacity to limit disease severity and transmission of VoCs as they arise and circulate.

As the COVID-19 pandemic continues, long-term immunity against SARS-CoV-2 will be globally important. Official weekly cases have not dropped below 2 million since September of 2020, and continued emergence of novel variants have created a moving target for our immune systems and public health alike. The temporal aspects of COVID-19 immunity, particularly from repeated vaccination and infection, are less well understood than short-term vaccine efficacy. In this study, we explore the impact of combined vaccination and infection, also known as hybrid immunity, and the timing thereof on the quality and quantity of antibodies produced by a cohort of 96 health care workers. We find robust neutralizing antibody responses among those with hybrid immunity against all variants, including Omicron BA.2, and we further found significantly improved neutralizing titers with longer vaccine-infection intervals up to 400 days. These results indicate that anti-SARS-CoV-2 antibody responses undergo continual maturation following primary exposure by either vaccination or infection for at least 400 days after last antigen exposure. We show that neutralizing antibody responses improved upon secondary boosting with greater impact seen after extended intervals. Our findings may also extend to booster vaccine doses, a critical consideration in future vaccine campaign strategies.

Sphingolipids are a critical family of membrane lipids with diverse functions in eukaryotic cells, and a growing body of literature supports that these lipids play essential roles during the lifecycles of viruses. While small molecule inhibitors of sphingolipid synthesis and metabolism are widely used, the advent of CRISPR-based genomic editing techniques allows for nuanced exploration into the manners in which sphingolipids influence various stages of viral infections. Here we describe some of these critical considerations needed in designing studies utilizing genomic editing techniques for manipulating the sphingolipid metabolic pathway, as well as the current body of literature regarding how viruses depend on the products of this pathway. Here, we highlight the ways in which sphingolipids affect viruses as these pathogens interact with and influence their host cell and describe some of the many open questions remaining in the field.

The capsid (CA) domain of the HIV-1 precursor Gag (PrGag) protein plays multiple roles in HIV-1 replication, and is central to the assembly of immature virions, and mature virus cores. CA proteins themselves are composed of N-terminal domains (NTDs) and C-terminal domains (CTDs). We have investigated the interactions of CA with anti-CA nanobodies, which derive from the antigen recognition regions of camelid heavy chain-only antibodies. The one CA NTD-specific and two CTD-specific nanobodies we analyzed proved sensitive and specific HIV-1 CA detection reagents in immunoassays. When co-expressed with HIV-1 Gag proteins in cells, the NTD-specific nanobody was efficiently assembled into virions and did not perturb virus assembly. In contrast, the two CTD-specific nanobodies reduced PrGag processing, virus release and HIV-1 infectivity. Our results demonstrate the feasibility of Gag-targeted nanobody inhibition of HIV-1.

Each novel SARS-CoV-2 variant renews concerns about decreased vaccine efficacy caused by evasion of vaccine induced neutralizing antibodies. However, accumulating epidemiological data show that while vaccine prevention of infection varies, protection from severe disease and death remains high. Thus, immune responses beyond neutralization could contribute to vaccine efficacy. Polyclonal antibodies function through their Fab domains that neutralize virus directly, and Fc domains that induce non-neutralizing host responses via engagement of Fc receptors on immune cells. To understand how vaccine induced neutralizing and non-neutralizing activities synergize to promote protection, we leverage sera from 51 SARS-CoV-2 uninfected health-care workers after two doses of the BNT162b2 mRNA vaccine. We show that BNT162b2 elicits antibodies that neutralize clinical isolates of wildtype and five variants of SARS-CoV-2, including Omicron BA.2, and, critically, induce Fc effector functions. FcγRIIIa/CD16 activity is linked to neutralizing activity and associated with post-translational afucosylation and sialylation of vaccine specific antibodies. Further, neutralizing and non-neutralizing functions diminish with age, with limited polyfunctional breadth, magnitude and coordination observed in those ≥65 years old compared to

Current coronavirus disease 2019 (COVID-19) vaccines effectively reduce overall morbidity and mortality and are vitally important to controlling the pandemic. Individuals who previously recovered from COVID-19 have enhanced immune responses after vaccination (hybrid immunity) compared with their naïve-vaccinated peers; however, the effects of post-vaccination breakthrough infections on humoral immune response remain to be determined. Here, we measure neutralizing antibody responses from 104 vaccinated individuals, including those with breakthrough infections, hybrid immunity, and no infection history. We find that human immune sera after breakthrough infection and vaccination after natural infection broadly neutralize SARS-CoV-2 (severe acute respiratory coronavirus 2) variants to a similar degree. Although age negatively correlates with antibody response after vaccination alone, no correlation with age was found in breakthrough or hybrid immune groups. Together, our data suggest that the additional antigen exposure from natural infection substantially boosts the quantity, quality, and breadth of humoral immune response regardless of whether it occurs before or after vaccination.

As a complement to vaccines, small-molecule therapeutic agents are needed to treat or prevent infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, which cause COVID-19. Affinity selection–mass spectrometry was used for the discovery of botanical ligands to the SARS-CoV-2 spike protein. Cannabinoid acids from hemp (Cannabis sativa) were found to be allosteric as well as orthosteric ligands with micromolar affinity for the spike protein. In follow-up virus neutralization assays, cannabigerolic acid and cannabidiolic acid prevented infection of human epithelial cells by a pseudovirus expressing the SARS-CoV-2 spike protein and prevented entry of live SARS-CoV-2 into cells. Importantly, cannabigerolic acid and cannabidiolic acid were equally effective against the SARS-CoV-2 alpha variant B.1.1.7 and the beta variant B.1.351. Orally bioavailable and with a long history of safe human use, these cannabinoids, isolated or in hemp extracts, have the potential to prevent as well as treat infection by SARS-CoV-2.

Background COVID-19 pandemic has a devastating impact on the economies and health care system of sub-Saharan Africa. Healthcare workers (HWs), the main actors of the health system, are at higher risk because of their occupation. Serology-based estimates of SARS-CoV-2 infection among HWs represent a measure of HWs’ exposure to the virus and could be used as a guide to the prevalence of SARS-CoV-2 in the community and valuable in combating COVID-19. This information is currently lacking in Ethiopia and other African countries. This study aimed to develop an in-house antibody testing assay, assess the prevalence of SARS-CoV-2 antibodies among Ethiopian high-risk frontline HWs. Methods We developed and validated an in-house Enzyme-Linked Immunosorbent Assay (ELISA) for specific detection of anti-SARS-CoV-2 receptor binding domain immunoglobin G (IgG) antibodies. We then used this assay to assess the seroprevalence among HWs in five public hospitals located in different geographic regions of Ethiopia. From consenting HWs, blood samples were collected between December 2020 and February 2021, the period between the two peaks of COVID-19 in Ethiopia. Socio-demographic and clinical data were collected using questionnaire-based interviews. Descriptive statistics and bivariate and multivariate logistic regression were used to determine the overall and post-stratified seroprevalence and the association between seropositivity and potential risk factors. Results Our successfully developed in-house assay sensitivity was 100% in serum samples collected 2- weeks after the first onset of symptoms whereas its specificity in pre-COVID-19 pandemic sera was 97.7%. Using this assay, we analyzed a total of 1997 sera collected from HWs. Of 1997 HWs who provided a blood sample, and demographic and clinical data, 51.7% were females, 74.0% had no symptoms compatible with COVID-19, and 29.0% had a history of contact with suspected or confirmed patients with SARS-CoV-2 infection. The overall seroprevalence was 39.6%. The lowest (24.5%) and the highest (48.0%) seroprevalence rates were found in Hiwot Fana Specialized Hospital in Harar and ALERT Hospital in Addis Ababa, respectively. Of the 821 seropositive HWs, 224(27.3%) of them had a history of symptoms consistent with COVID-19 while 436 (> 53%) of them had no contact with COVID-19 cases as well as no history of COVID-19 like symptoms. A history of close contact with suspected/confirmed COVID-19 cases is associated with seropositivity (Adjusted Odds Ratio (AOR) = 1.4, 95% CI 1.1–1.8; p = 0.015). Conclusion High SARS-CoV-2 seroprevalence levels were observed in the five Ethiopian hospitals. These findings highlight the significant burden of asymptomatic infection in Ethiopia and may reflect the scale of transmission in the general population.

The spread of the vaccine-resistant Omicron severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants threatens unvaccinated and fully vaccinated individuals, and accelerated booster vaccination campaigns are underway to mitigate the ongoing wave of Omicron cases. The immunity provided by standard vaccine regimens, boosted regimens, and immune responses elicited by vaccination plus natural infection remain incompletely understood. The magnitude, quality, and durability of serological responses, and the likelihood of protection against future SARS-CoV-2 variants following these modes of exposure, are poorly characterized but are critical to the future trajectory of the coronavirus disease 2019 (COVID-19) pandemic.Ninety-nine individuals were semi-randomly selected from a larger vaccination cohort following vaccination and, in some cases, breakthrough infection. We analyzed spike receptor-binding domain-specific immunoglobulin G (IgG), IgA, and IgM by enzyme-linked immunosorbent assay, neutralizing antibody titers against live SARS-CoV-2 variants, and antibody-dependent cell-mediated phagocytosis.In 99 vaccinated adults, compared with responses after two doses of an mRNA regimen, the immune responses 3 months after a third vaccine dose and 1 month after breakthrough infection due to prior variants show dramatic increases in magnitude, potency, and breadth, including increased antibody-dependent cellular phagocytosis and robust neutralization of the currently circulating Omicron BA.2 variant.Boosters and natural infection substantially boost immune responses. As the number of Omicron sub-variant cases rise and as global vaccination and booster campaigns continue, an increasing proportion of the world's population will acquire potent immune responses that may be protective against future SARS-CoV-2 variants.This work was funded by the M. J. Murdock Charitable Trust, the OHSU Foundation, the NIH (T32HL083808), and OHSU Innovative IDEA.

BackgroundCOVID-19 pandemic has a devastating impact on the economies and health care system of sub-Saharan Africa. Healthcare workers (HWs), the main actors of the health system, are at higher-risk because of their occupation. Serology-based estimates of SARS-CoV-2 infection among HWs represent a measure of HWs’ exposure to the virus and a guide to the prevalence of SARS-CoV-2 in the community. This information is currently lacking in Ethiopia and other African countries. This study aimed to develop an in-house antibody testing assay, assess the prevalence of SARS-CoV-2 antibodies among Ethiopian high-risk frontline HWs.Methods and findingsA cross-sectional seroprevalence study was conducted among HWs in five public hospitals located in different geographic regions of Ethiopia. Socio-demographic and clinical data were collected using questionnaire-based interviews. From consenting HWs, blood samples were collected between December 2020 and February 2021, the period between the two peaks of COVID-19 in Ethiopia. The collected sera were tested using an in-house immunoglobin G (IgG) enzyme-linked immunosorbent assay (ELISA) for SARS-CoV-2 specific antibodies on sera collected from HWs. Of 1,997 HWs who provided a blood sample, demographic and clinical data, 50.5% were female, 74.0% had no symptoms compatible with COVID-19, and 29.0% had history of contact with suspected or confirmed patient with SARS-CoV-2 infection. The overall seroprevalence was 39.6%. The lowest (24.5%) and the highest (48.0%) seroprevalence rates were found in Hiwot Fana Specialized Hospital in Harar and ALERT Hospital in Addis Ababa, respectively. Of the 821 seropositive HWs, 224(27.3%) had history of symptoms consistent with COVID-19. A history of close contact with suspected/confirmed COVID-19 cases was strongly associated with seropositivity (Adjusted odds Ratio (AOR) =1.4, 95% CI 1.1-1.8; p=0.015).ConclusionHigh SARS-CoV-2 seroprevalence levels were observed in the five Ethiopian hospitals. These findings highlight the significant burden of asymptomatic infection in Ethiopia, and may reflect the scale of transmission in the general population.Author summaryWhy was this study done?Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global public health threat, including AfricaThe actual morbidity and mortality associated with SARS-CoV-2 infection in Ethiopia underestimated due to the limited molecular testing capacity.We have limited knowledge about the seroprevalence of COVID-19 among health workers in Ethiopia.This study aimed to develop an in-house immunoglobin G (IgG) enzyme-linked immunosorbent assay (ELISA) for SARS-CoV-2 specific antibodies on sera collected from HWs and to find out the proportion of healthcare workers who have developed antibodies specific to SARS-CoV-2 from five public hospitals located in the different regions of Ethiopia.What did the researchers do and find?A cross-sectional seroprevalence study was conducted among HWs in five public hospitals located in different geographic regions of Ethiopia.Socio-demographic and clinical data were collected from recruited and consented participants using questionnaire-based interviews.Blood samples were collected from participants between December 2020 and February 2021, the period between the two peaks of COVID-19 in Ethiopia.The collected sera were tested using an in-house ELISA for SARS-CoV-2 specific antibodies on sera collected from HWs.Approximately 40% of the 1,997 healthcare workers who participated in this study had antibodies against SARS-CoV-2 infection.No association between seropositivity and study participants’ age, gender, occupation, and comorbid medical conditions.What do these findings mean?The observed high seroprevalence among healthcare workers regardless of their occupation suggests the cryptic but massive SARS-CoV-2 transmission in urban hospital settings.Most of the seropositive healthcare workers in the present study were asymptomatic, and might pose a threat to the most vulnerable populations such as individuals with comorbid medical conditions.Given the low level of vaccine roll-out (1%), this study highlights the need to strengthen health workers’ adherence to personal protection practices such as wearing face masks to protect individuals at high risk of developing severe COVID-19 illness after SARS-CoV-2 infection.

The unprecedented severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has called for substantial investigations into the capacity of the human immune system to protect against reinfection and keep pace with the evolution of SARS-CoV-2. We evaluated the magnitude and durability of the SARS-CoV-2-specific antibody responses against parental WA-1 SARS-CoV-2 receptor-binding domain (RBD) and a representative variant of concern (VoC) RBD using antibodies from 2 antibody compartments: long-lived plasma cell-derived plasma antibodies and antibodies encoded by SARS-CoV-2-specific memory B cells (MBCs). Thirty-five participants naturally infected with SARS-CoV-2 were evaluated; although only 25 of 35 participants had VoC RBD-reactive plasma antibodies, 34 of 35 (97%) participants had VoC RBD-reactive MBC-derived antibodies. Our finding that 97% of previously infected individuals have MBCs specific for variant RBDs provides reason for optimism regarding the capacity of vaccination, prior infection, and/or both, to elicit immunity with the capacity to limit disease severity and transmission of VoCs as they arise and circulate.

Access to fast and reliable nucleic acid testing continues to play a key role in controlling the COVID-19 pandemic, especially in the context of increased vaccine break-through risks due to new variants. We report a rapid, low-cost (~ 2 USD), simple-to-use nucleic acid test kit for self-administered at-home testing without lab instrumentation. The entire sample-to-answer workflow takes

We compared the serum neutralizing antibody titers before and after two doses of the BNT162b2 COVID-19 vaccine in ten individuals who recovered from SARS-CoV-2 infection prior to vaccination to 20 individuals with no history of infection, against clinical isolates of B.1.1.7, B.1.351, P.1, and the original SARS-CoV-2 virus. Vaccination boosted pre-existing levels of anti-SARS-CoV-2 spike antibodies 10-fold in previously infected individuals, but not to levels significantly higher than those of uninfected vaccinees. However, neutralizing antibody titers increased in previously infected vaccinees relative to uninfected vaccinees against every variant tested: 5.2-fold against B.1.1.7, 6.5-fold against B.1.351, 4.3-fold against P.1, and 3.4-fold against original SARS-CoV-2. Our study indicates that a first-generation COVID-19 vaccine provides broad protection from SARS-CoV-2variants in individuals with previous infection.

We tested human sera from large, demographically balanced cohorts of BNT162b2 vaccine recipients (n=51) and COVID-19 patients (n=44) for neutralizing antibodies against SARS-CoV-2 variants B.1.1.7 and B.1.351. Although the effect is more pronounced in the vaccine cohort, both B.1.1.7 and B.1.351 show significantly reduced levels of neutralization by vaccinated and convalescent sera. Age is negatively correlated with neutralization in vaccinee, and levels of variant-specific RBD antibodies are proportional to neutralizing activities.

Mycobacterium tuberculosis (Mtb) invades alveolar macrophages through phagocytosis to establish infection and cause disease. The molecular mechanisms underlying Mtb entry are still poorly understood., Phagocytosis by alveolar macrophages is the obligate first step in Mycobacterium tuberculosis (Mtb) infection, yet the mechanism underlying this process is incompletely understood. Here, we show that Mtb invasion relies on an intact sphingolipid biosynthetic pathway. Inhibition or knockout of early sphingolipid biosynthetic enzymes greatly reduces Mtb uptake across multiple phagocytic cell types without affecting other forms of endocytosis. While the phagocytic receptor dectin-1 undergoes normal clustering at the pathogen contact sites, sphingolipid biosynthetic mutant cells fail to segregate the regulatory phosphatase CD45 from the clustered receptors. Blocking sphingolipid production also impairs downstream activation of Rho GTPases, actin dynamics, and phosphoinositide turnover at the nascent phagocytic cup. Moreover, we found that production of sphingomyelin, not glycosphingolipids, is essential for Mtb uptake. Collectively, our data support a critical role of sphingomyelin biosynthesis in an early stage of Mtb infection and provide novel insights into the mechanism underlying phagocytic entry of this pathogen.

The lipid composition of HIV-1 virions is enriched in sphingomyelin (SM), but the roles that SM or other sphingolipids (SLs) might play in the HIV-1 replication pathway have not been elucidated. In human cells, SL levels are regulated by ceramide synthase (CerS) enzymes that produce ceramides, which can be converted to SMs, hexosylceramides, and other SLs. In many cell types, CerS2, which catalyzes the synthesis of very long chain ceramides, is the major CerS. We have examined how CerS2 deficiency affects the assembly and infectivity of HIV-1. As expected, we observed that very long chain ceramide, hexosylceramide, and SM were reduced in CerS2 knockout cells. CerS2 deficiency did not affect HIV-1 assembly or the incorporation of the HIV-1 envelope (Env) protein into virus particles, but it reduced the infectivites of viruses produced in the CerS2-deficient cells. The reduced viral infection levels were dependent on HIV-1 Env, since HIV-1 particles that were pseudotyped with the vesicular stomatitis virus glycoprotein did not exhibit reductions in infectivity. Moreover, cell–cell fusion assays demonstrated that the functional defect of HIV-1 Env in CerS2-deficient cells was independent of other viral proteins. Overall, our results indicate that the altered lipid composition of CerS2-deficient cells specifically inhibit the HIV-1 Env receptor binding and/or fusion processes.

Mucosal-associated invariant T (MAIT) cells are an innate-like T cell subset important in the early response to bacterial and viral lung pathogens. MAIT cells recognize bacterial small molecule metabolites presented on the Class I-like molecule MR1. As with other Class I and Class II molecules, MR1 can likely sample ligands in the intracellular environment through multiple cellular pathways. Rab6, a small GTPase that regulates a number of endosomal trafficking pathways including retrograde transport to the trans-Golgi network (TGN), is involved in the presentation of ligands from Mycobacterium tuberculosis (Mtb) to MAIT cells. The Rab6-mediated trafficking pathway contains endosomal compartments that share features with the Mtb intracellular compartment. Using inducible expression of MR1, this study demonstrates that Rab6 regulates the recycling of MR1 molecules from the cell surface through endosomal trafficking compartments to the TGN. This Rab6-dependent pool of recycled MR1, which is available for reloading with ligands from bacterial pathogens like Mtb, may be important for early recognition of infected cells by MAIT cells in the lung.

Current COVID-19 vaccines significantly reduce overall morbidity and mortality and are vitally important to halting the pandemic; individuals who previously recovered from COVID-19 display enhanced immune responses after vaccination (hybrid immunity) compared to their naïve-vaccinated peers. However, the effects of vaccine breakthrough infections on humoral immune response remain to be determined. Here, we measure neutralizing antibody responses from 104 vaccinated individuals including those with breakthrough infections, hybrid immunity, and no infection history. We find that human immune sera collected after either a breakthrough infection or a vaccination post-natural infection will both broadly neutralize SARS-CoV-2 variants to a similar degree. While age negatively correlates with antibody response after vaccination alone, no such association was found in breakthrough or hybrid immune groups. Together, our data suggest that the additional antigen exposure from natural infection substantially boosts the quantity, quality, and breadth of humoral immune response regardless of whether it occurs before or after vaccination. Supported by grants from the M.J. Murdock Charitable Trust, OHSU Innovative IDEA grant (1018784), NIH (R01AI145835, T32HL083808), and an unrestricted grant from the OHSU Foundation.

Mycobacterium tuberculosis (Mtb) is a pathogen with a massive global health impact, and yet fundamental questions remain regarding the underlying mechanisms of its entry and subversion of innate immune mechanisms – particularly the manners in which host lipids influence the course of an infection. This pathogen gains entry to host alveolar macrophages via phagocytosis and, within the phagosome, Mtb perturbs host lipid metabolism and distribution. Here, we've shown that Mtb relies on an intact sphingolipid biosynthesis pathway to gain entry to host cells but that sphingolipids restrict the intracellular growth of this pathogen. We used genetic ablation and small molecule inhibition to perturb sphingolipid biosynthesis, resulting in reduced Mtb uptake across multiple phagocytic cell types without affecting receptor mediated endocytosis. Mechanistically, we visualized the dynamics of several signaling events during phagocytosis to demonstrate that sphingolipid deficiency results in poor initiation of phagocytic signaling. Intriguingly, sphingolipid inhibition at later stages of infection may result in enhanced intracellular Mtb growth and accelerated lysosomal damage and subsequent phagolysosomal escape. This study has revealed that the sphingolipid family may constitute a double-edged sword which plays multiple roles during Mtb infection. In understanding the interplay between Mycobacterium tuberculosis and host factors such as sphingolipids, we hope to identify novel methods of treatment for this ancient and global pathogen. Supported by grants from NIH (R21 3R21AI124225-01A1 and R01 1R01AI14154 9-01A1)

Zika virus (ZIKV) is a re-emergent flavivirus that triggered a global health emergency from 2015 to 2017. ZIKV and other impactful flaviviruses, including dengue virus, lack the antiviral treatments and vaccines needed to diminish their threat to communities world-wide. This is in part due to gaps in our understanding of the flavivirus lifecycle; despite numerous investigations into flavivirus non-structural (NS) protein function. I hope to forge a deeper understanding of crucial NS protein functions and interactions throughout the ZIKV replication cycle. I will use alpaca-derived variable heavy-chain-only (VHH) antibody fragments, isolated from an alpaca immunized with ZIKV NS proteins, to perturb the replication cycle and therefore identify key NS protein epitopes used during replication. I individually purified NS1, NS2b, NS3, NS4b, NS5 for use in alpaca immunization, phage display panning, and enzyme-linked immunosorbent assays (ELISAs). From the alpaca peripheral blood I isolated RNA and amplified VHH genes, enriched for NS-protein binders with iterative phage display, and screened potential high binders via ELISA. I have isolated VHH sequences that interact with the ZIKV helicase, NS3, and the RNA-dependent RNA polymerase, NS5. I plan to purify each VHH and express them in ZIKA-permissive A549 cells as stable lines. Using purified VHH, I will analyze their binding ability in-vitro by ELISA and bio layer interferometry, and quantify in-vitro inhibition of NS3 protease and helicase activity for NS3 binders, and NS5 polymerase and methyltransferase activity. With the stable cells lines I will investigate each VHHs ability to impede ZIKV virus production and formation of replication complexes in infected cell lines. Support from training grant "Interactions at the Microbe-Host Interface" T32AI007472

This study of fully vaccinated health care workers examines antibody levels and variant cross-neutralization after COVID-19 breakthrough infection.

This study examines the relationship between age and neutralizing antibody titers against the SARS-CoV-2 USA-WA1/2020 strain and the P.1 variant after 2 doses of the BNT162b2 vaccine.

The central role of myeloid cells in driving autoimmune diseases and cancer has raised interest in manipulating their function or depleting them for therapeutic benefits. To achieve this, antibodies are used to antagonize differentiation, survival and polarization signals or to kill target cells, for example in the form of antibody-drug conjugates (ADC). The action of ADC in vivo can be hard to predict based on target expression pattern alone. The biology of the targeted receptor as well as its interplay with the ADC can have drastic effects on cell apoptosis versus survival. Here we investigated the efficacy of CD11b or Ly-6C/Ly-6G-specific variable fragments of camelid heavy chain-only antibodies (VHH) conjugated to Pseudomonas exotoxin A to deplete myeloid cells in vitro and in vivo. Our data highlight striking differences in cell killing in vivo, depending on the cell subset and organs targeted, but not antigen expression level or VHH affinity. We observed striking differences in depletion efficiency of monocytes versus granulocytes in mice. Despite similar binding of Ly-6C/Ly-6G-specific VHH immunotoxin to granulocytes and monocytes, granulocytes were significantly more sensitive than monocytes to immunotoxins treatment. Our results illustrate the need of early, thorough in vivo characterization of ADC candidates.

Zika virus (ZIKV), an arbovirus of global concern, remodels intracellular membranes to form replication sites. How ZIKV dysregulates lipid networks to allow this, and consequences for disease, is poorly understood. Here, we perform comprehensive lipidomics to create a lipid network map during ZIKV infection. We find that ZIKV significantly alters host lipid composition, with the most striking changes seen within subclasses of sphingolipids. Ectopic expression of ZIKV NS4B protein results in similar changes, demonstrating a role for NS4B in modulating sphingolipid pathways. Disruption of sphingolipid biosynthesis in various cell types, including human neural progenitor cells, blocks ZIKV infection. Additionally, the sphingolipid ceramide redistributes to ZIKV replication sites, and increasing ceramide levels by multiple pathways sensitizes cells to ZIKV infection. Thus, we identify a sphingolipid metabolic network with a critical role in ZIKV replication and show that ceramide flux is a key mediator of ZIKV infection., Zika virus (ZIKV) remodels intracellular membranes for replication, but the role of different lipid types for infection and disease is unclear. Here, the authors perform lipidomics, show perturbation of the lipid network during ZIKV infection and show that ceramides are important for ZIKV infection.

Phagocytosis by phagocytes such as neutrophils is a crucial part of the host innate immune response against invading pathogens. Phagocytosis is a complex process that initiates with the binding of the particles on the cell surface of the phagocytes through the interaction of pattern recognition receptors with ligands on the surface of the pathogens. During this process, phagocytes undergo extensive membrane reorganization and cytoskeleton rearrangement at their cell surface. To gain better insight about the molecular mechanisms of this dynamic cellular process, visualization and quantification in a high-throughput manner is essential. Here, we describe a microscope-based method to visualize and quantify phagocytic uptake of pathogens (such as bacteria and fungi) and model particulates that are larger than 0.5 μm (such as Zymosan A and IgG-coated beads).

Phagocytosis by phagocytes such as neutrophils is a crucial part of the host innate immune response against invading pathogens. Phagocytosis is a complex process that initiates with the binding of the particles on the cell surface of the phagocytes through the interaction of pattern recognition receptors with ligands on the surface of the pathogens. During this process, phagocytes undergo extensive membrane reorganization and cytoskeleton rearrangement at their cell surface. To gain better insight about the molecular mechanisms of this dynamic cellular process, visualization and quantification in a high-throughput manner is essential. Here, we describe a microscope-based method to visualize and quantify phagocytic uptake of pathogens (such as bacteria and fungi) and model particulates that are larger than 0.5 μm (such as Zymosan A and IgG-coated beads).

Mucosal Associated Invariant T (MAIT) cells can sense intracellular infection by a broad array of pathogens. These cells are activated upon encountering microbial antigen(s) displayed by MR1 on the surface of an infected cell. Human MR1 undergoes alternative splicing. The full length isoform, MR1A, can activate MAIT cells, while the function of the isoforms, MR1B and MR1C, are not well characterized.In this report, we sought to characterize these splice variants. Using a transcriptomic analysis in conjunction with qPCR, we find that that MR1A and MR1B transcripts are widely expressed. Despite the widespread expression of MR1A and MR1B, only MR1A can present mycobacterial antigen to MAIT cells. Coexpression of MR1B with MR1A serves to decrease MAIT cell activation following bacterial infection. However, expression of MR1B prior to MR1A lowers total MR1A abundance, suggesting competition between MR1A and MR1B for either ligands or chaperones required for folding and/or trafficking. Finally, we evaluated CD4/CD8 double positive thymocytes expressing surface MR1. Relative MR1A/MR1B expression in MR1-expressing thymocytes is associated with their prevalence.Our results suggest alternative splicing of MR1 represents a means of regulating MAIT activation in response to microbial ligand.FundingThis work was supported by NIH T32HL083808 (EK, GAN, EM), VA Merit Award I01CX001562 (MJH), NIH R01AI29976 (MJH), NIH R01AI048090 (DML), NIH R21AI124225-01A1 (FT) and VA Merit Award I01BX000533 (DML). The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.

Variability in bacterial sterilization is a key feature of Mycobacterium tuberculosis (Mtb) disease. In a population of human macrophages, there are macrophages that restrict Mtb growth and those that do not. However, the sources of heterogeneity in macrophage state during Mtb infection are poorly understood. Here, we perform RNAseq on restrictive and permissive macrophages and reveal that the expression of genes involved in GM-CSF signaling discriminates between the two subpopulations. We demonstrate that blocking GM-CSF makes macrophages more permissive of Mtb growth while addition of GM-CSF increases bacterial control. In parallel, we find that the loss of bacterial control that occurs in HIV-Mtb coinfected macrophages correlates with reduced GM-CSF secretion. Treatment of coinfected cells with GM-CSF restores bacterial control. Thus, we leverage the natural variation in macrophage control of Mtb to identify a critical cytokine response for regulating Mtb survival and identify components of the antimicrobial response induced by GM-CSF., GM-CSF has been implicated in Mycobacterium tuberculosis (Mtb) host control. Here, the authors show that activation of GM-CSF discriminates Mtb-restrictive from -permissive macrophages while co-infection with HIV disrupts GM-CSF-mediated signaling, rendering macrophages susceptible to bacterial infection.

Tuberculosis, the pathological result of infection by Mycobacterium tuberculosis (M.tb), represents a massive and persistent global health burden with vast overrepresentation in regions with poor access to healthcare. This pathogen survives intracellularly within lung macrophages, and has been shown to extensively reprogram the host lipid compartment to the effect of enhanced survival and immune evasion. Sphingolipids are a class of lipid with extensive functions in a wide variety of cellular contexts, including apoptosis, autophagy, stress response, and inflammation. There remains a significant knowledge gap surrounding the lipid-mediated interactions between M.tb and its host. Our investigation aims to explore the mechanisms through which M.tb harnesses host-derived sphingolipids to gain entry to the host and to survive intracellularly. To this end, we use chemical and genetic perturbations of the sphingolipid de novo synthesis pathway and fluorescence microscopy to show that sphingolipids are required for efficient phagocytosis of M.tb. We furthermore show that the dynamics of phosphoinositides at the phagosomal synapse are perturbed upon blockade of sphingolipid synthesis. Finally, we demonstrate that perturbing the homeostasis of a specific sphingolipid (sphingomyelin) through both enrichment and depletion at the cell surface reduces M.tb uptake. This work represents a first step in building comprehensive knowledge of the mechanisms of host-pathogen interactions in M.tb, and demonstrates a cohort of methodologies that may be applied to a variety of pathogenic contexts.

In the ongoing coronavirus disease 2019 (COVID-19) pandemic, there remain unanswered questions regarding the nature and significance of the humoral immune response toward other coronavirus infections. Here, we investigate the cross-reactivity of antibodies raised against the first severe acute respiratory syndrome coronavirus (SARS-CoV) for their reactivity toward SARS-CoV-2. We extensively characterize a selection of 10 antibodies covering all of the SARS-CoV structural proteins: spike, membrane, nucleocapsid, and envelope. Although nearly all of the examined SARS-CoV antibodies display some level of reactivity to SARS-CoV-2, we find only partial cross-neutralization for the spike antibodies. The implications of our work are two-fold. First, we establish a set of antibodies with known reactivity to both SARS-CoV and SARS-CoV-2, which will allow further study of both viruses. Second, we provide empirical evidence of the high propensity for antibody cross-reactivity between distinct strains of human coronaviruses, which is critical information for designing diagnostic and vaccine strategies for COVID-19., Graphical Abstract, Bates et al. extensively examine the cross-reactivity of selected monoclonal antibodies raised against SARS-CoV structural proteins with SARS-CoV-2. Although most SARS-CoV antibodies display remarkable cross-reactivity toward SARS-CoV-2 proteins, the spike antibodies of SARS-CoV show minimal cross-neutralization of live SARS-CoV-2 virus.

Author(s): Tafesse, Fikadu G; Leier, Hans C; Weinstein, Jules B; Navratil, Aaron R; Messer, William B; Dennis, Edward A; Barklis, Eric