Long-term in vitro exposure of Treponema pallidum to sub-bactericidal doxycycline did not induce resistance: Implications for doxy-PEP and syphilis.

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.

Safety and Efficacy of SAB-185 for Non-hospitalized Adults with COVID-19: A Randomized Clinical Trial.

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.

Age-dependent heterogeneity in the antigenic effects of mutations to influenza hemagglutinin.

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.

Genomic epidemiology and evolution of rhinovirus in western Washington State, 2021-22.

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.

The kinetics and durability of antibody and T-cell responses to SARS-CoV-2 in children.

BACKGROUND: The kinetics and durability of T-cell responses to SARS-CoV-2 in children are not well-characterized. We studied a cohort of children aged 6 months to 20 years with COVID-19 in whom peripheral blood mononuclear cells (PBMC) and sera were archived at approximately 1, 6, and 12 months post-symptom onset. METHODS: We compared antibody (N = 85) and T-cell responses (N = 26) to nucleocapsid (N) and spike (S) glycoprotein over time across four age strata: 6 months to 5 years, 5-9, 10-14, and 15-20 years. RESULTS: N-specific antibody responses declined over time, becoming undetectable in 26/32 (81%) children by approximately one year post-infection. Functional breadth of anti-N CD4+ T-cell responses also declined over time and were positively correlated with N-antibody responses (Pearson's r = 0.31, p = 0.008). CD4+ T-cell responses to S displayed greater functional breadth than N in unvaccinated children, and, along with neutralization titers, were stable over time and similar across age strata. Functional profiles of CD4+ T-cell responses against S were not significantly modulated by vaccination. CONCLUSIONS: Our data reveal durable, age-independent T-cell immunity to SARS-CoV-2 structural proteins in children over time following COVID-19 infection as well as S-Ab responses overall, in comparison to declining antibody responses to N.

CMV Reactivation during Pre-Transplantation Evaluation: A Novel Risk Factor for Post-Transplantation CMV Reactivation.

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.

Functional and structural basis of human parainfluenza virus type 3 neutralization with human monoclonal antibodies.

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.

A neutralizing antibody prevents postfusion transition of measles virus fusion protein.

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.

Ocular Mpox in a Breastfeeding Healthcare Provider.

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.

Fine-scale spatial and social patterns of SARS-CoV-2 transmission from identical pathogen sequences.

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.

Bright New Resources for Syphilis Research: Genetically Encoded Fluorescent Tags for Treponema pallidum and Sf1Ep Cells.

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.

Treponema pallidum Periplasmic and Membrane Proteins Are Recognized by Circulating and Skin CD4+ T Cells.

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.

Efficacy of late-onset antiviral treatment in immune-compromised hosts with persistent SARS-CoV-2 infection.

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.

Viral genomic variation and the severity of genital HSV-2 infection as quantified by shedding rate: a viral genome-wide association study.

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.

Proceedings of the Clinical Microbiology Open 2023: discussions about pandemic preparedness.

The 4th Clinical Microbiology Open (CMO) took place in Carlsbad, California, on 10 and 11 February 2023. This event facilitated discussion between clinical and public health laboratory directors, government agencies, and industry representatives from the companies that make up ASM's Corporate Council. While many topics were discussed, much of the discussion focused on pandemic preparedness. There were four major questions addressed: (i) When is the perfect the enemy of good in pandemic testing? (ii) What other types of pathogens might cause another pandemic and how would this affect laboratory response? (iii) What research is needed to better understand the effectiveness of the pandemic response? (iv) What have we learned about the utility of self and at-home testing in future pandemics? This review serves as a summary of these discussions.

Characterization of treatment resistance and viral kinetics in the setting of single- versus dual-active monoclonal antibodies against SARS-CoV-2.

BACKGROUND: Monoclonal antibodies (mAbs) represent a crucial antiviral strategy for SARS-CoV-2 infection, but it is unclear whether combination mAbs offer a benefit over single-active mAb treatment. Amubarvimab and romlusevimab significantly reduced the risk of hospitalizations or death in the ACTIV-2/A5401 trial. Certain SARS-CoV-2 variants are intrinsically resistant against romlusevimab, leading to only single-active mAb therapy with amubarvimab in these variants. We evaluated virologic outcomes in individuals treated with single- versus dual-active mAbs. METHODS: Participants were non-hospitalized adults at higher risk of clinical progression randomized to amubarvimab plus romlusevimab or placebo. Quantitative SARS-CoV-2 RNA levels and targeted S gene next-generation sequencing was performed on anterior nasal samples. We compared viral load kinetics and resistance emergence between individuals treated with effective single- versus dual-active mAbs depending on the infecting variant. RESULTS: Study participants receiving single- and dual-active mAbs had similar demographics, baseline nasal viral load, symptom score, and symptom duration. Compared to single-active mAb, treatment with dual-active mAbs led to faster viral load decline at study day 3 (p < 0.001) and day 7 (p < 0.01). Treatment-emergent resistance mutations were more likely to be detected after amubarvimab plus romlusevimab treatment than placebo (2.6% vs 0%, P < 0.001), and more frequently detected in the setting of single-active compared to dual-active mAb treatment (7.2% vs 1.1%, p < 0.01). Single-active and dual-active mAb treatment resulted in similar decrease in rates of hospitalizations or death. CONCLUSION: Compared to single-active mAb therapy, dual-active mAbs led to similar clinical outcomes, but significantly faster viral load decline and a lower risk of emergent resistance.

The effect of gastrointestinal graft-versus-host disease and diarrhea on the pharmacokinetic profile of sotrovimab in hematopoietic stem cell transplant recipients.

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.