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Science is humanity's best insurance against threats from nature, but it is a fragile enterprise that must be nourished and protected. The preponderance of scientific evidence indicates a natural origin for SARS-CoV-2. Yet, the theory that SARS-CoV-2 was engineered in and escaped from a lab dominates media attention, even in the absence of strong evidence. We discuss how the resulting anti-science movement puts the research community, scientific research, and pandemic preparedness at risk.
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The recently discovered methodologies to cultivate and genetically manipulate Treponema pallidum subsp. pallidum (T. pallidum) have significantly helped syphilis research, allowing the in vitro evaluation of antibiotic efficacy, performance of controlled studies to assess differential treponemal gene expression, and generation of loss-of-function mutants to evaluate the contribution of specific genetic loci to T. pallidum virulence. Building on this progress, we engineered the T. pallidum SS14 strain to express a red-shifted green fluorescent protein (GFP) and Sf1Ep cells to express mCherry and blue fluorescent protein (BFP) for enhanced visualization. These new resources improve microscopy- and cell sorting-based applications for T. pallidum, better capturing the physical interaction between the host and pathogen, among other possibilities. Continued efforts to develop and share new tools and resources are required to help our overall knowledge of T. pallidum biology and syphilis pathogenesis reach that of other bacterial pathogens, including spirochetes.
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BACKGROUND: Human rhinoviruses (RV) primarily cause the common cold, but infection outcomes vary from subclinical to severe cases, including asthma exacerbations and fatal pneumonia in immunocompromised individuals. To date, therapeutic strategies have been hindered by the high diversity of serotypes. Global surveillance efforts have traditionally focused on sequencing VP1 or VP2/VP4 genetic regions, leaving gaps in our understanding of RV genomic diversity. METHODS: We sequenced 1,078 RV genomes from nasal swabs of symptomatic and asymptomatic individuals to explore viral evolution during two epidemiologically distinct periods in Washington State: when the COVID-19 pandemic affected the circulation of other seasonal respiratory viruses except for RV (February - July 2021), and when the seasonal viruses reemerged with the severe RSV and influenza outbreak (November-December 2022). We constructed maximum likelihood and BEAST-phylodynamic trees to characterize intra-genotype evolution. RESULTS: We detected 99 of 168 known genotypes and observed inter-genotypic recombination and genotype cluster swapping from 2021 to 2022. We found a significant association between the presence of symptoms and viral load, but not with RV species or genotype. Phylodynamic trees, polyprotein selection pressure, and Shannon entropy revealed co-circulation of divergent clades within genotypes with high amino acid constraints throughout polyprotein. DISCUSSION: Our study underscores the dynamic nature of RV genomic epidemiology within a localized geographic region, as more than 20% of existing genotypes within each RV species co-circulated each studied month. Our findings also emphasize the importance of investigating correlations between rhinovirus genotypes and serotypes to understand long-term immunity and cross-protection.
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Filoviruses are some of the most lethal viruses in the modern world, and increasing numbers of filovirus species and genera have been discovered in recent years. Despite the potential severity of filovirus outbreaks in the human population, comparably few sensitive pan-filovirus RT-PCR assays have been described that might facilitate early detection and prevention. Here, we present a new pan-filovirus RT-PCR assay targeting the L polymerase gene for detection of all known mammalian filoviruses. We demonstrate the detection of 10 synthetic filovirus RNA templates with analytical sensitivity ranging from 178 to 3,354 copies/mL, without cross-reactivity on 10 non-filoviral human viral species. We verified assay performance on 10 inactivated filovirus isolates, yielding initial sensitivities of 0.012-44.17 TCID50/mL. We coupled this broadly reactive RT-PCR with a deep sequencing workflow that is amenable to high-throughput pooling to maximize detection and discovery potential. In summary, this pan-filovirus RT-PCR assay targets the most conserved filovirus gene, offers the widest breadth of coverage to date, and may help in the detection and discovery of novel filoviruses.IMPORTANCEFiloviruses remain some of the most mysterious viruses known to the world, with extremely high lethality rates and significant pandemic potential. Yet comparably few filovirus species and genera have been discovered to date and questions surround the definitive host species for zoonotic infections. Here, we describe a novel broadly reactive RT-PCR assay targeting the conserved L polymerase gene for high-throughput screening for filoviruses in a variety of clinical and environmental specimens. We demonstrate the assay can detect all known mammalian filoviruses and determine the sensitivity and specificity of the assay on synthetic RNA sequences, inactivated filovirus isolates, and non-filoviral species.
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Doxycycline post-exposure prophylaxis (doxy-PEP) could significantly reduce syphilis incidence. However, the increase in intermittent doxycycline usage might select resistant Treponema pallidum (T. pallidum) strains. To assess whether resistance to doxycycline could be induced in this pathogen, we exposed the SS14 strain in vitro both intermittently and continuously to a sub-bactericidal doxycycline concentration that still exerts antibiotic pressure. During and after each exposure experiment, we assessed the doxycycline minimal inhibitory concentration in test and control treponemes and performed whole genome sequencing, concluding that no resistance developed. This work suggests that doxycycline-resistant T. pallidum is not an immediate threat for doxy-PEP implementation.
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Doxycycline post-exposure prophylaxis (doxy-PEP) could significantly reduce syphilis incidence. However, the increase in intermittent doxycycline usage might select resistant Treponema pallidum ( T. pallidum ) strains. To assess whether resistance to doxycycline could be induced in this pathogen, we exposed the SS14 strain in vitro both intermittently and continuously to a sub-bactericidal doxycycline concentration that still exerts antibiotic pressure. During and after each exposure experiment, we assessed the doxycycline minimal inhibitory concentration in test and control treponemes and performed whole genome sequencing, concluding that no resistance developed. This work suggests that doxycycline-resistant T. pallidum is not an immediate threat for doxy-PEP implementation.
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Human influenza virus evolves to escape neutralization by polyclonal antibodies. However, we have a limited understanding of how the antigenic effects of viral mutations vary across the human population and how this heterogeneity affects virus evolution. Here, we use deep mutational scanning to map how mutations to the hemagglutinin (HA) proteins of two H3N2 strains, A/Hong Kong/45/2019 and A/Perth/16/2009, affect neutralization by serum from individuals of a variety of ages. The effects of HA mutations on serum neutralization differ across age groups in ways that can be partially rationalized in terms of exposure histories. Mutations that were fixed in influenza variants after 2020 cause greater escape from sera from younger individuals compared with adults. Overall, these results demonstrate that influenza faces distinct antigenic selection regimes from different age groups and suggest approaches to understand how this heterogeneous selection shapes viral evolution.
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Anticorpos Antivirais , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Vírus da Influenza A Subtipo H3N2 , Influenza Humana , Mutação , Humanos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Adulto , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Influenza Humana/virologia , Influenza Humana/imunologia , Fatores Etários , Pessoa de Meia-Idade , Adulto Jovem , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Antígenos Virais/genética , Antígenos Virais/imunologia , Adolescente , Evolução Molecular , Idoso , CriançaRESUMO
BACKGROUND: To address the need for novel COVID-19 therapies, we evaluated the fully-human polyclonal antibody product SAB-185 in a phase 3 clinical trial. METHODS: Non-hospitalized high-risk adults within 7 days of COVID-19 symptom onset were randomized 1:1 to open-label SAB-185 3,840 units/kg or casirivimab/imdevimab 1200 mg. Non-inferiority comparison was undertaken for the pre-Omicron population (casirivimab/imdevimab expected to be fully active) and superiority comparison for the Omicron population (casirivimab/imdevimab not expected to be active). Primary outcomes were the composite of all-cause hospitalizations/deaths and grade ≥3 treatment-emergent adverse events (TEAEs) through day 28. Secondary outcomes included time to sustained symptom improvement and resolution. RESULTS: Enrollment was terminated early due to low hospitalization/death rates upon Omicron emergence. 733 adults were randomized, 255 included in pre-Omicron and 392 in Omicron analysis populations. Hospitalizations/deaths occurred in 6 (5.0%) and 3 (2.2%) of pre-Omicron SAB-185 and casirivimab/imdevimab arms, respectively (absolute difference [95% CI] 2.7% [-2.3%, 8.6%]), inconclusive for non-inferiority; and 5 (2.5%) versus 3 (1.5%) (absolute difference 1.0% [-2.3%, 4.5%]) for Omicron. Risk ratios for grade ≥3 TEAEs were 0.94 [0.52, 1.71] (pre-Omicron) and 1.71 [0.96, 3.07] (Omicron). Time to symptom improvement and resolution were shorter for SAB-185, median 11 vs 14 (pre-Omicron) and 11 vs 13 days (Omicron) (symptom improvement), and 16 vs 24 days and 18 vs >25 days (symptom resolution), p<0.05 for symptom resolution for Omicron only. CONCLUSIONS: SAB-185 had an acceptable safety profile with faster symptom resolution in the Omicron population. Additional studies are needed to characterize its efficacy for COVID-19.
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Cytomegalovirus (CMV) disease occurs occasionally before allogeneic hematopoietic cell transplantation (HCT) and is associated with poor post-HCT outcomes; however, the impact of pre-HCT CMV reactivation is unknown. Pre-HCT CMV reactivation was assessed in HCT candidates from the preemptive antiviral therapy (2007-17) and letermovir prophylaxis (2018-21) eras. CMV DNA PCR surveillance was routinely performed during the pre-HCT work-up period, and antiviral therapy was recommended according to risk for progression to CMV disease. Risk factors for pre-HCT CMV reactivation were characterized and the associations of pre-HCT CMV reactivation with post-HCT outcomes were examined using logistic regression and Cox proportional hazard models, respectively. A total of 1694 patients were identified and 11% had pre-HCT CMV reactivation 14 days (median; IQR 6-23 days) before HCT. Lymphopenia (≤300 cells/uL) was the strongest risk factor for pre-HCT CMV reactivation at multiple PCR levels. In the preemptive therapy era, patients with pre-HCT CMV reactivation had a significantly increased risk of CMV reactivation by day 100 as well as CMV disease and death by 1 year post-HCT. Clearance of pre-HCT CMV reactivation was associated with a lower risk of post-HCT CMV reactivation. Similar associations with post-HCT CMV endpoints were observed in a cohort of patients receiving letermovir prophylaxis. Pre-HCT CMV reactivation can be routinely detected in high-risk HCT candidates and is a significant risk factor for post-HCT CMV reactivation and disease. Pre-HCT CMV DNA PCR surveillance is recommended in high-risk HCT candidates and antiviral therapy may be indicated to prevent post-HCT CMV reactivation.
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A healthcare provider unknowingly treated a patient with mpox and subsequently developed ocular mpox without rash. She breastfed during illness; her infant was not infected. This report addresses 3 challenges in mpox management and control: diagnosis in the absence of rash, exposures in healthcare settings, and management of lactating patients.
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Pathogen genomics can provide insights into disease transmission patterns, but new methods are needed to handle modern large-scale pathogen genome datasets. Genetically proximal viruses indicate epidemiological linkage and are informative about transmission events. Here, we leverage pairs of identical sequences using 114,298 SARS-CoV-2 genomes collected via sentinel surveillance from March 2021 to December 2022 in Washington State, USA, with linked age and residence information to characterize fine-scale transmission. The location of pairs of identical sequences is highly consistent with expectations from mobility and social contact data. Outliers in the relationship between genetic and mobility data can be explained by SARS-CoV-2 transmission between postal codes with male prisons, consistent with transmission between prison facilities. Transmission patterns between age groups vary across spatial scales. Finally, we use the timing of sequence collection to understand the age groups driving transmission. This work improves our ability to characterize transmission from large pathogen genome datasets.
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Measles virus (MeV) presents a public health threat that is escalating as vaccine coverage in the general population declines and as populations of immunocompromised individuals, who cannot be vaccinated, increase. There are no approved therapeutics for MeV. Neutralizing antibodies targeting viral fusion are one potential therapeutic approach but have not yet been structurally characterized or advanced to clinical use. We present cryo-electron microscopy (cryo-EM) structures of prefusion F alone [2.1-angstrom (Å) resolution], F complexed with a fusion-inhibitory peptide (2.3-Å resolution), F complexed with the neutralizing and protective monoclonal antibody (mAb) 77 (2.6-Å resolution), and an additional structure of postfusion F (2.7-Å resolution). In vitro assays and examination of additional EM classes show that mAb 77 binds prefusion F, arrests F in an intermediate state, and prevents transition to the postfusion conformation. These structures shed light on antibody-mediated neutralization that involves arrest of fusion proteins in an intermediate state.
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Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Microscopia Crioeletrônica , Vírus do Sarampo , Proteínas Virais de Fusão , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/química , Vírus do Sarampo/imunologia , Vírus do Sarampo/química , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/química , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/química , Humanos , Conformação ProteicaAssuntos
Antibacterianos , Azitromicina , Farmacorresistência Bacteriana , Macrolídeos , Sífilis , Treponema pallidum , Humanos , Antibacterianos/farmacologia , Antibacterianos/provisão & distribuição , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana/genética , Macrolídeos/farmacologia , Macrolídeos/uso terapêutico , América do Norte , Sífilis/tratamento farmacológico , Sífilis/genética , Sífilis/microbiologia , Treponema pallidum/efeitos dos fármacos , Treponema pallidum/genética , Treponema pallidum/isolamento & purificação , Azitromicina/farmacologia , Azitromicina/uso terapêutico , Estados Unidos , Canadá , Penicilina G Benzatina/farmacologia , Penicilina G Benzatina/provisão & distribuição , Penicilina G Benzatina/uso terapêutico , Doxiciclina/farmacologia , Doxiciclina/provisão & distribuição , Doxiciclina/uso terapêuticoRESUMO
The recently discovered methodologies to cultivate and genetically manipulate Treponema pallidum subsp. pallidum ( T. pallidum ) have significantly helped syphilis research, allowing the in vitro evaluation of antibiotic efficacy, performance of controlled studies to assess differential treponemal gene expression, and generation of loss-of-function mutants to evaluate the contribution of specific genetic loci to T. pallidum virulence. Building on this progress, we engineered the T. pallidum SS14 strain to express a red-shifted Green Fluorescent Protein (GFP) and Sf1Ep cells to express mCherry and blue fluorescent protein (BFP) for enhanced visualization. These new resources improve microscopy- and cell sorting-based applications for T. pallidum , better capturing the physical interaction between the host and pathogen, among other possibilities. Continued efforts to develop and share new tools and resources are required to help our overall knowledge of T. pallidum biology and syphilis pathogenesis reach that of other bacterial pathogens, including spirochetes. Graphical abstract: By employing genetic engineering, T. pallidum was modified to express GFP, and Sf1Ep cells to express mCherry on the cytoplasmic membrane and BFP in the nucleus. These new resources for syphilis research will facilitate experimental designs to better define the complex interplay between T. pallidum and the host during infection.
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Human parainfluenza virus type 3 (hPIV3) is a respiratory pathogen that can cause severe disease in older people and infants. Currently, vaccines against hPIV3 are in clinical trials but none have been approved yet. The haemagglutinin-neuraminidase (HN) and fusion (F) surface glycoproteins of hPIV3 are major antigenic determinants. Here we describe naturally occurring potently neutralizing human antibodies directed against both surface glycoproteins of hPIV3. We isolated seven neutralizing HN-reactive antibodies and a pre-fusion conformation F-reactive antibody from human memory B cells. One HN-binding monoclonal antibody (mAb), designated PIV3-23, exhibited functional attributes including haemagglutination and neuraminidase inhibition. We also delineated the structural basis of neutralization for two HN and one F mAbs. MAbs that neutralized hPIV3 in vitro protected against infection and disease in vivo in a cotton rat model of hPIV3 infection, suggesting correlates of protection for hPIV3 and the potential clinical utility of these mAbs.
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Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Proteína HN , Vírus da Parainfluenza 3 Humana , Infecções por Respirovirus , Sigmodontinae , Proteínas Virais de Fusão , Animais , Vírus da Parainfluenza 3 Humana/imunologia , Vírus da Parainfluenza 3 Humana/genética , Humanos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/química , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/química , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/química , Proteína HN/imunologia , Proteína HN/química , Proteína HN/genética , Infecções por Respirovirus/imunologia , Infecções por Respirovirus/virologia , Modelos Animais de Doenças , Testes de Neutralização , Linfócitos B/imunologia , Modelos MolecularesRESUMO
BACKGROUND: Histologic and serologic studies suggest the induction of local and systemic Treponema pallidum-specific CD4+ T-cell responses to T. pallidum infection. We hypothesized that T. pallidum-specific CD4+ T cells are detectable in blood and in the skin rash of secondary syphilis and persist in both compartments after treatment. METHODS: Peripheral blood mononuclear cells collected from 67 participants were screened by interferon-γ (IFN-γ) ELISPOT response to T. pallidum sonicate. T. pallidum-reactive T-cell lines from blood and skin were probed for responses to 89 recombinant T. pallidum antigens. Peptide epitopes and HLA class II restriction were defined for selected antigens. RESULTS: We detected CD4+ T-cell responses to T. pallidum sonicate ex vivo. Using T. pallidum-reactive T-cell lines we observed recognition of 14 discrete proteins, 13 of which localize to bacterial membranes or the periplasmic space. After therapy, T. pallidum-specific T cells persisted for at least 6 months in skin and 10 years in blood. CONCLUSIONS: T. pallidum infection elicits an antigen-specific CD4+ T-cell response in blood and skin. T. pallidum-specific CD4+ T cells persist as memory in both compartments long after curative therapy. The T. pallidum antigenic targets we identified may be high-priority vaccine candidates.
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Linfócitos T CD4-Positivos , Pele , Sífilis , Treponema pallidum , Humanos , Treponema pallidum/imunologia , Linfócitos T CD4-Positivos/imunologia , Sífilis/imunologia , Pele/imunologia , Pele/microbiologia , Adulto , Masculino , Feminino , Proteínas de Membrana/imunologia , Antígenos de Bactérias/imunologia , Pessoa de Meia-Idade , Interferon gama/metabolismo , Proteínas de Bactérias/imunologia , ELISPOT , Leucócitos Mononucleares/imunologia , Adulto JovemRESUMO
The immunocompromised are at high risk of prolonged SARS-CoV-2 infection and progression to severe COVID-19. However, efficacy of late-onset direct-acting antiviral (DAA) therapy with therapeutics in clinical use and experimental drugs to mitigate persistent viral replication is unclear. In this study, we employed an immunocompromised mouse model, which supports prolonged replication of SARS-CoV-2 to explore late-onset treatment options. Tandem immuno-depletion of CD4 + and CD8 + T cells in C57BL/6 mice followed by infection with SARS-CoV-2 variant of concern (VOC) beta B.1.351 resulted in prolonged infection with virus replication for five weeks after inoculation. Early-onset treatment with nirmatrelvir/ritonavir (paxlovid) or molnupiravir was only moderately efficacious, whereas the experimental therapeutic 4'-fluorourdine (4'-FlU, EIDD-2749) significantly reduced virus load in upper and lower respiratory compartments four days post infection (dpi). All antivirals significantly lowered virus burden in a 7-day treatment regimen initiated 14 dpi, but paxlovid-treated animals experienced rebound virus replication in the upper respiratory tract seven days after treatment end. Viral RNA was detectable 28 dpi in paxlovid-treated animals, albeit not in the molnupiravir or 4'-FlU groups, when treatment was initiated 14 dpi and continued for 14 days. Low-level virus replication continued 35 dpi in animals receiving vehicle but had ceased in all treatment groups. These data indicate that late-onset DAA therapy significantly shortens the duration of persistent virus replication in an immunocompromised host, which may have implications for clinical use of antiviral therapeutics to alleviate the risk of progression to severe disease in highly vulnerable patients. Importance: Four years after the onset of the global COVID-19 pandemic, the immunocompromised are at greatest risk of developing life-threatening severe disease. However, specific treatment plans for this most vulnerable patient group have not yet been developed. Employing a CD4 + and CD8 + T cell-depleted immunocompromised mouse model of SARS-CoV-2 infection, we explored therapeutic options of persistent infections with standard-of-care paxlovid, molnupiravir, and the experimental therapeutic 4'-FlU. Late-onset treatment initiated 14 days after infection was efficacious, but only 4'-FlU was rapidly sterilizing. No treatment-experienced viral variants with reduced susceptibility to the drugs emerged, albeit virus replication rebounded in animals of the paxlovid group after treatment end. This study supports the use of direct-acting antivirals for late-onset management of persistent SARS-CoV-2 infection in immunocompromised hosts. However, treatment courses likely require to be extended for maximal therapeutic benefit, calling for appropriately powered clinical trials to meet the specific needs of this patient group.
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BACKGROUND: The clinical severity of genital HSV-2 infection varies widely among infected persons with some experiencing frequent genital lesions while others are asymptomatic. The viral genital shedding rate is closely associated with and has been established as a surrogate marker of clinical severity. METHODS: To assess the relationship between viral genetics and shedding, we assembled a set of 145 persons who had the severity of their genital herpes quantified through determination of their HSV genital shedding rate. An HSV-2 sample from each person was sequenced and biallelic variants among these genomes were identified. RESULTS: We found no association between metrics of genome-wide variation in HSV-2 and shedding rate. A viral genome-wide association study (vGWAS) identified the minor alleles of three individual unlinked variants as significantly associated with higher shedding rate (p<8.4x10-5): C44973T (A512T), a non-synonymous variant in UL22 (glycoprotein H); A74534G, a synonymous variant in UL36 (large tegument protein); and T119283C, an intergenic variant. We also found an association between the total number of minor alleles for the significant variants and shedding rate (p=6.6x10-7). CONCLUSIONS: These results add to a growing body of literature for HSV suggesting a connection between viral genetic variation and clinically important phenotypes of infection.
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BACKGROUND: Monoclonal antibodies (mAbs) are utilized broadly to treat cancer and infectious diseases, and mAb exposure (serum concentration over time) is one predictor of overall treatment efficacy. Herein, we present findings from a clinical trial evaluating the pharmacokinetics (PK) of the long-acting mAb sotrovimab targeting SARS-CoV-2 in hematopoietic cell transplant (HCT) recipients. METHODS: All participants received an intravenous infusion of sotrovimab within one week prior to initiating the pre-transplant preparative regimen. The serum concentration of sotrovimab was measured longitudinally for up to 24 weeks post-transplant. RESULTS: Compared to non-HCT participants, we found that mAb clearance was 10% and 26% higher in autologous and allogeneic HCT recipients, respectively. Overall sotrovimab exposure was approximately 15% lower in HCT recipients compared to non-HCT recipients. Exposure was significantly reduced in HCT recipients who developed diarrhea and lower gastrointestinal (GI) graft-versus-host disease (GVHD) post-transplant. CONCLUSIONS: These data show that sotrovimab exposure may be reduced in HCT recipients, possibly related to increased GI clearance in patients with GVHD. This phenomenon has implications for dose selection and duration of efficacy with sotrovimab and potentially other mAbs in this vulnerable patient population. Thus, mAb dose regimens developed in non-HCT populations may have to be optimized when applied to HCT populations.