Emerging SARS-CoV-2 Resistance After Antiviral Treatment.

Importance: Previous studies have identified mutations in SARS-CoV-2 strains that confer resistance to nirmatrelvir, yet how often this resistance arises and its association with posttreatment virologic rebound is not well understood. Objective: To examine the prevalence of emergent antiviral resistance after nirmatrelvir treatment and its association with virologic rebound. Design, Setting, and Participants: This cohort study enrolled outpatient adults with acute COVID-19 infection from May 2021 to October 2023. Participants were divided into those who received antiviral therapy and those who did not. The study was conducted at a multicenter health care system in Boston, Massachusetts. Exposure: Treatment regimen, including none, nirmatrelvir, and remdesivir. Main Outcomes and Measures: The primary outcome was emergent SARS-CoV-2 antiviral resistance, defined as the detection of antiviral resistance mutations, which were not present at baseline, were previously associated with decreased antiviral efficacy, and emerged during or after completion of a participant's treatment. Next-generation sequencing was used to detect low frequency mutations down to 1% of the total viral population. Results: Overall, 156 participants (114 female [73.1%]; median [IQR] age, 56 [38-69] years) were included. Compared with 63 untreated individuals, the 79 who received nirmatrelvir were older and more commonly immunosuppressed. After sequencing viral RNA from participants' anterior nasal swabs, nirmatrelvir resistance mutations were detected in 9 individuals who received nirmatrelvir (11.4%) compared with 2 of those who did not (3.2%) (P = .09). Among the individuals treated with nirmatrelvir, those who were immunosuppressed had the highest frequency of resistance emergence (5 of 22 [22.7%]), significantly greater than untreated individuals (2 of 63 [3.1%]) (P = .01). Similar rates of nirmatrelvir resistance were found in those who had virologic rebound (3 of 23 [13.0%]) vs those who did not (6 of 56 [10.7%]) (P = .86). Most of these mutations (10 of 11 [90.9%]) were detected at low frequencies (<20% of viral population) and reverted to the wild type at subsequent time points. Emerging remdesivir resistance mutations were only detected in immunosuppressed individuals (2 of 14 [14.3%]) but were similarly low frequency and transient. Global Initiative on Sharing All Influenza Data analysis showed no evidence of increased nirmatrelvir resistance in the United States after the authorization of nirmatrelvir. Conclusions and Relevance: In this cohort study of 156 participants, treatment-emergent nirmatrelvir resistance mutations were commonly detected, especially in individuals who were immunosuppressed. However, these mutations were generally present at low frequencies and were transient in nature, suggesting a low risk for the spread of nirmatrelvir resistance in the community with the current variants and drug usage patterns.

Pathogenesis of Post-Tuberculosis Lung Disease: Defining Knowledge Gaps and Research Priorities at the 2nd International Post-Tuberculosis Symposium.

Post-tuberculosis (TB) lung disease (PTLD) is increasingly recognized as a major contributor to the global burden of chronic lung disease, with recent estimates indicating that over half of TB survivors have impaired lung function after successful completion of TB treatment. However, the pathologic mechanisms that contribute to PTLD are not well understood, thus limiting the development of therapeutic interventions to improve long-term outcomes after TB. This report summarizes the work of the "Pathogenesis and Risk Factors Committee" for the Second International Post-Tuberculosis Symposium, which took place in Stellenbosch, South Africa in April 2023. The committee first identified six areas with high translational potential: (1) tissue matrix destruction, including the role of matrix metalloproteinase dysregulation and neutrophil activity, (2) fibroblasts and profibrotic activity, (3) granuloma fate and cell death pathways, (4) mycobacterial factors including pathogen burden, (5) animal models, and (6) the impact of key clinical risk factors including HIV, diabetes, smoking, malnutrition, and alcohol. We share here the key findings from a literature review of those areas, highlighting knowledge gaps and areas where further research is needed.

Postinfectious Pulmonary Complications: Establishing Research Priorities to Advance the Field: An Official American Thoracic Society Workshop Report.

Continued improvements in the treatment of pulmonary infections have paradoxically resulted in a growing challenge of individuals with postinfectious pulmonary complications (PIPCs). PIPCs have been long recognized after tuberculosis, but recent experiences such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic have underscored the importance of PIPCs following other lower respiratory tract infections. Independent of the causative pathogen, most available studies of pulmonary infections focus on short-term outcomes rather than long-term morbidity among survivors. In this document, we establish a conceptual scope for PIPCs with discussion of globally significant pulmonary pathogens and an examination of how these pathogens can damage different components of the lung, resulting in a spectrum of PIPCs. We also review potential mechanisms for the transition from acute infection to PIPC, including the interplay between pathogen-mediated injury and aberrant host responses, which together result in PIPCs. Finally, we identify cross-cutting research priorities for the field to facilitate future studies to establish the incidence of PIPCs, define common mechanisms, identify therapeutic strategies, and ultimately reduce the burden of morbidity in survivors of pulmonary infections.

Rapid, antibiotic incubation-free determination of tuberculosis drug resistance using machine learning and Raman spectroscopy.

Tuberculosis (TB) is the world's deadliest infectious disease, with over 1.5 million deaths and 10 million new cases reported anually. The causative organism Mycobacterium tuberculosis (Mtb) can take nearly 40 d to culture, a required step to determine the pathogen's antibiotic susceptibility. Both rapid identification and rapid antibiotic susceptibility testing of Mtb are essential for effective patient treatment and combating antimicrobial resistance. Here, we demonstrate a rapid, culture-free, and antibiotic incubation-free drug susceptibility test for TB using Raman spectroscopy and machine learning. We collect few-to-single-cell Raman spectra from over 25,000 cells of the Mtb complex strain Bacillus Calmette-Guérin (BCG) resistant to one of the four mainstay anti-TB drugs, isoniazid, rifampicin, moxifloxacin, and amikacin, as well as a pan-susceptible wildtype strain. By training a neural network on this data, we classify the antibiotic resistance profile of each strain, both on dried samples and on patient sputum samples. On dried samples, we achieve >98% resistant versus susceptible classification accuracy across all five BCG strains. In patient sputum samples, we achieve ~79% average classification accuracy. We develop a feature recognition algorithm in order to verify that our machine learning model is using biologically relevant spectral features to assess the resistance profiles of our mycobacterial strains. Finally, we demonstrate how this approach can be deployed in resource-limited settings by developing a low-cost, portable Raman microscope that costs <$5,000. We show how this instrument and our machine learning model enable combined microscopy and spectroscopy for accurate few-to-single-cell drug susceptibility testing of BCG.

A mouse model of TB-associated lung fibrosis reveals persistent inflammatory macrophage populations during treatment.

Post-TB lung disease (PTLD) causes a significant burden of global disease. Fibrosis is a central component of many clinical features of PTLD. To date, we have a limited understanding of the mechanisms of TB-associated fibrosis and how these mechanisms are similar to or dissimilar from other fibrotic lung pathologies. We have adapted a mouse model of TB infection to facilitate the mechanistic study of TB-associated lung fibrosis. We find that the morphologies of fibrosis that develop in the mouse model are similar to the morphologies of fibrosis observed in human tissue samples. Using Second Harmonic Generation (SHG) microscopy, we are able to quantify a major component of fibrosis, fibrillar collagen, over time and with treatment. Inflammatory macrophage subpopulations persist during treatment; matrix remodeling enzymes and inflammatory gene signatures remain elevated. Our mouse model suggests that there is a therapeutic window during which adjunctive therapies could change matrix remodeling or inflammatory drivers of tissue pathology to improve functional outcomes after treatment for TB infection.

Evasion of NKG2D-mediated cytotoxic immunity by sarbecoviruses.

SARS-CoV-2 and other sarbecoviruses continue to threaten humanity, highlighting the need to characterize common mechanisms of viral immune evasion for pandemic preparedness. Cytotoxic lymphocytes are vital for antiviral immunity and express NKG2D, an activating receptor conserved among mammals that recognizes infection-induced stress ligands (e.g., MIC-A/B). We found that SARS-CoV-2 evades NKG2D recognition by surface downregulation of MIC-A/B via shedding, observed in human lung tissue and COVID-19 patient serum. Systematic testing of SARS-CoV-2 proteins revealed that ORF6, an accessory protein uniquely conserved among sarbecoviruses, was responsible for MIC-A/B downregulation via shedding. Further investigation demonstrated that natural killer (NK) cells efficiently killed SARS-CoV-2-infected cells and limited viral spread. However, inhibition of MIC-A/B shedding with a monoclonal antibody, 7C6, further enhanced NK-cell activity toward SARS-CoV-2-infected cells. Our findings unveil a strategy employed by SARS-CoV-2 to evade cytotoxic immunity, identify the culprit immunevasin shared among sarbecoviruses, and suggest a potential novel antiviral immunotherapy.

Enhancing antibody responses by multivalent antigen display on thymus-independent DNA origami scaffolds.

Protein-based virus-like particles (P-VLPs) are commonly used to spatially organize antigens and enhance humoral immunity through multivalent antigen display. However, P-VLPs are thymus-dependent antigens that are themselves immunogenic and can induce B cell responses that may neutralize the platform. Here, we investigate thymus-independent DNA origami as an alternative material for multivalent antigen display using the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, the primary target of neutralizing antibody responses. Sequential immunization of mice with DNA-based VLPs (DNA-VLPs) elicits protective neutralizing antibodies to SARS-CoV-2 in a manner that depends on the valency of the antigen displayed and on T cell help. Importantly, the immune sera do not contain boosted, class-switched antibodies against the DNA scaffold, in contrast to P-VLPs that elicit strong B cell memory against both the target antigen and the scaffold. Thus, DNA-VLPs enhance target antigen immunogenicity without generating scaffold-directed immunity and thereby offer an important alternative material for particulate vaccine design.

SARS-CoV-2 viral clearance and evolution varies by type and severity of immunodeficiency.

Despite vaccination and antiviral therapies, immunocompromised individuals are at risk for prolonged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but the immune defects that predispose an individual to persistent coronavirus disease 2019 (COVID-19) remain incompletely understood. In this study, we performed detailed viro-immunologic analyses of a prospective cohort of participants with COVID-19. The median times to nasal viral RNA and culture clearance in individuals with severe immunosuppression due to hematologic malignancy or transplant (S-HT) were 72 and 40 days, respectively, both of which were significantly longer than clearance rates in individuals with severe immunosuppression due to autoimmunity or B cell deficiency (S-A), individuals with nonsevere immunodeficiency, and nonimmunocompromised groups (P < 0.01). Participants who were severely immunocompromised had greater SARS-CoV-2 evolution and a higher risk of developing resistance against therapeutic monoclonal antibodies. Both S-HT and S-A participants had diminished SARS-CoV-2-specific humoral responses, whereas only the S-HT group had reduced T cell-mediated responses. This highlights the varied risk of persistent COVID-19 across distinct immunosuppressive conditions and suggests that suppression of both B and T cell responses results in the highest contributing risk of persistent infection.

Rapid, antibiotic incubation-free determination of tuberculosis drug resistance using machine learning and Raman spectroscopy.

Tuberculosis (TB) is the world's deadliest infectious disease, with over 1.5 million deaths annually and 10 million new cases reported each year. The causative organism, Mycobacterium tuberculosis (Mtb) can take nearly 40 days to culture, a required step to determine the pathogen's antibiotic susceptibility. Both rapid identification of Mtb and rapid antibiotic susceptibility testing (AST) are essential for effective patient treatment and combating antimicrobial resistance. Here, we demonstrate a rapid, culture-free, and antibiotic incubation-free drug susceptibility test for TB using Raman spectroscopy and machine learning. We collect few-to-single-cell Raman spectra from over 25,000 cells of the MtB complex strain Bacillus Calmette Guerin (BCG) resistant to one of the four mainstay anti-TB drugs, isoniazid, rifampicin, moxifloxacin and amikacin, as well as a pan susceptible wildtype strain. By training a neural network on this data, we classify the antibiotic resistance profile of each strain, both on dried samples and in patient sputum samples. On dried samples, we achieve >98% resistant versus susceptible classification accuracy across all 5 BCG strains. In patient sputum samples, we achieve ~79% average classification accuracy. We develop a feature recognition algorithm in order to verify that our machine learning model is using biologically relevant spectral features to assess the resistance profiles of our mycobacterial strains. Finally, we demonstrate how this approach can be deployed in resource-limited settings by developing a low-cost, portable Raman microscope that costs <$5000. We show how this instrument and our machine learning model enables combined microscopy and spectroscopy for accurate few-to-single-cell drug susceptibility testing of BCG.

SARS-CoV-2 Virologic Rebound With Nirmatrelvir-Ritonavir Therapy : An Observational Study.

BACKGROUND: Data are conflicting regarding an association between treatment of acute COVID-19 with nirmatrelvir-ritonavir (N-R) and virologic rebound (VR). OBJECTIVE: To compare the frequency of VR in patients with and without N-R treatment for acute COVID-19. DESIGN: Observational cohort study. SETTING: Multicenter health care system in Boston, Massachusetts. PARTICIPANTS: Ambulatory adults with acute COVID-19 with and without use of N-R. INTERVENTION: Receipt of 5 days of N-R treatment versus no COVID-19 therapy. MEASUREMENTS: The primary outcome was VR, defined as either a positive SARS-CoV-2 viral culture result after a prior negative result or 2 consecutive viral loads above 4.0 log10 copies/mL that were also at least 1.0 log10 copies/mL higher than a prior viral load below 4.0 log10 copies/mL. RESULTS: Compared with untreated persons (n = 55), those taking N-R (n = 72) were older, received more COVID-19 vaccinations, and more commonly had immunosuppression. Fifteen participants (20.8%) taking N-R had VR versus 1 (1.8%) who was untreated (absolute difference, 19.0 percentage points [95% CI, 9.0 to 29.0 percentage points]; P = 0.001). All persons with VR had a positive viral culture result after a prior negative result. In multivariable models, only N-R use was associated with VR (adjusted odds ratio, 10.02 [CI, 1.13 to 88.74]; P = 0.038). Virologic rebound was more common among those who started therapy within 2 days of symptom onset (26.3%) than among those who started 2 or more days after symptom onset (0%) (P = 0.030). Among participants receiving N-R, those who had VR had prolonged shedding of replication-competent virus compared with those who did not have VR (median, 14 vs. 3 days). Eight of 16 participants (50% [CI, 25% to 75%]) with VR also reported symptom rebound; 2 were completely asymptomatic. No post-VR resistance mutations were detected. LIMITATIONS: Observational study design with differences between the treated and untreated groups; positive viral culture result was used as a surrogate marker for risk for ongoing viral transmission. CONCLUSION: Virologic rebound occurred in approximately 1 in 5 people taking N-R, often without symptom rebound, and was associated with shedding of replication-competent virus. PRIMARY FUNDING SOURCE: National Institutes of Health.

Vps18 contributes to phagosome membrane integrity in Mycobacterium tuberculosis-infected macrophages.

Mycobacterium tuberculosis (Mtb) has evolved to be exquisitely adapted to survive within host macrophages. The capacity to damage the phagosomal membrane has emerged as central to Mtb virulence. While Mtb factors driving membrane damage have been described, host factors that repair that damage to contain the pathogen remain largely unknown. We used a genome-wide CRISPR screen to identify novel host factors required to repair Mtb-damaged phagosomal membranes. Vacuolar protein sorting-associated protein 18 (Vps18), a member of the HOPS and CORVET trafficking complexes, was among the top hits. Vps18 colocalized with Mtb in macrophages beginning shortly after infection, and Vps18-knockout macrophages demonstrated increased damage of Mtb-containing phagosomes without impaired autophagy. Mtb grew more robustly in Vps18-knockout cells, and the first-line anti-tuberculosis antibiotic pyrazinamide was less effective. Our results identify Vps18 as required for phagosomal membrane integrity in Mtb-infected cells and suggest that modulating phagosome integrity may hold promise for improving the efficacy of antibiotic treatment for TB.

SARS-CoV-2 Viral Clearance and Evolution Varies by Extent of Immunodeficiency.

Despite vaccination and antiviral therapies, immunocompromised individuals are at risk for prolonged SARS-CoV-2 infection, but the immune defects that predispose to persistent COVID-19 remain incompletely understood. In this study, we performed detailed viro-immunologic analyses of a prospective cohort of participants with COVID-19. The median time to nasal viral RNA and culture clearance in the severe hematologic malignancy/transplant group (S-HT) were 72 and 40 days, respectively, which were significantly longer than clearance rates in the severe autoimmune/B-cell deficient (S-A), non-severe, and non-immunocompromised groups (P<0.001). Participants who were severely immunocompromised had greater SARS-CoV-2 evolution and a higher risk of developing antiviral treatment resistance. Both S-HT and S-A participants had diminished SARS-CoV-2-specific humoral, while only the S-HT group had reduced T cell-mediated responses. This highlights the varied risk of persistent COVID-19 across immunosuppressive conditions and suggests that suppression of both B and T cell responses results in the highest contributing risk of persistent infection.

Anamnestic humoral correlates of immunity across SARS-CoV-2 variants of concern.

While immune correlates against SARS-CoV-2 are typically defined at peak immunogenicity following vaccination, immunologic responses that expand selectively during the anamnestic response following infection can provide mechanistic and detailed insights into the immune mechanisms of protection. Moreover, whether anamnestic correlates are conserved across variants of concern (VOC), including the Delta and more distant Omicron VOC, remains unclear. To define the anamnestic correlates of immunity, across VOCs, we deeply profiled the humoral immune response in individuals infected with sequence-confirmed Delta or Omicron VOC after completing the vaccination series. While limited acute N-terminal domain and receptor-binding domain (RBD)-specific immune expansion was observed following breakthrough infection, a significant immunodominant expansion of opsonophagocytic Spike-specific antibody responses focused largely on the conserved S2-domain of SARS-CoV-2 was observed. This S2-specific functional humoral response continued to evolve over 2-3 weeks following Delta or Omicron breakthrough, targeting multiple VOCs and common coronaviruses. Strong responses were observed on the fusion peptide (FP) region and the heptad repeat 1 (HR1) region adjacent to the RBD. Notably, the FP is highly conserved across SARS-related coronaviruses and even non-SARS-related betacoronavirus. Taken together, our results point to a critical role of highly conserved, functional S2-specific responses in the anamnestic antibody response to SARS-CoV-2 infection across VOCs. These humoral responses linked to virus clearance can guide next-generation vaccine-boosting approaches to confer broad protection against future SARS-related coronaviruses. IMPORTANCE The Spike protein of SARS-CoV-2 is the primary target of antibody-based recognition. Selective pressures, be it the adaption to human-to-human transmission or evasion of previously acquired immunity, have spurred the emergence of variants of the virus such as the Delta and Omicron lineages. Therefore, understanding how antibody responses are expanded in breakthrough cases of previously vaccinated individuals can provide insights into key correlates of protection against current and future variants. Here, we show that vaccinated individuals who had documented COVID-19 breakthrough showed anamnestic antibody expansions targeting the conserved S2 subdomain of Spike, particularly within the fusion peptide region. These S2-directed antibodies were highly leveraged for non-neutralizing, phagocytic functions and were similarly expanded independent of the variant. We propose that through deep profiling of anamnestic antibody responses in breakthrough cases, we can identify antigen targets susceptible to novel monoclonal antibody therapy or vaccination-boosting strategies.

TLR2 is non-redundant in the population and subpopulation responses to Mycobacterium tuberculosis in macrophages and in vivo.

Tuberculosis (TB), caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb), is a global health threat. Targeting host pathways that modulate protective or harmful components of inflammation has been proposed as a therapeutic strategy that could aid sterilization or mitigate TB-associated permanent tissue damage. In purified form, many Mtb components can activate innate immune pathways. However, knowledge of the pathways that contribute most to the observed response to live Mtb is incomplete, limiting the possibility of precise intervention. We took a systematic, unbiased approach to define the pathways that drive the earliest immune response to Mtb. Using a macrophage model of infection, we compared the bulk transcriptional response to infection with the response to a panel of Mtb-derived putative innate immune ligands. We identified two axes of response: an NF-kB-dependent response similarly elicited by all Mtb pathogen-associated molecular patterns (PAMPs) and a type I interferon axis unique to cells infected with live Mtb. Consistent with growing literature data pointing to TLR2 as a dominant Mtb-associated PAMP, the TLR2 ligand PIM6 most closely approximated the NF-kB-dependent response to the intact bacterium. Quantitatively, the macrophage response to Mtb was slower and weaker than the response to purified PIM6. On a subpopulation level, the TLR2-dependent response was heterogeneously induced, with only a subset of infected cells expressing key inflammatory genes known to contribute to the control of infection. Despite potential redundancies in Mtb ligand/innate immune receptor interactions during in vivo infection, loss of the TLR2/PIM6 interaction impacted the cellular composition of both the innate and adaptive compartments. IMPORTANCE Tuberculosis (TB) is a leading cause of death globally. Drug resistance is outpacing new antibiotic discovery, and even after successful treatment, individuals are often left with permanent lung damage from the negative consequences of inflammation. Targeting host inflammatory pathways has been proposed as an approach that could either improve sterilization or improve post-treatment lung health. However, our understanding of the inflammatory pathways triggered by Mycobacterium tuberculosis (Mtb) in infected cells and lungs is incomplete, in part because of the complex array of potential molecular interactions between bacterium and host. Here, we take an unbiased approach to identify the pathways most central to the host response to Mtb. We examine how individual pathways are triggered differently by purified Mtb products or infection with the live bacterium and consider how these pathways inform the emergence of subpopulation responses in cell culture and in infected mice. Understanding how individual interactions and immune pathways contribute to inflammation in TB opens the door to the possibility of developing precise therapeutic interventions.

SARS-CoV-2 virologic rebound with nirmatrelvir-ritonavir therapy.

Objective: To compare the frequency of replication-competent virologic rebound with and without nirmatrelvir-ritonavir treatment for acute COVID-19. Secondary aims were to estimate the validity of symptoms to detect rebound and the incidence of emergent nirmatrelvir-resistance mutations after rebound. Design: Observational cohort study. Setting: Multicenter healthcare system in Boston, Massachusetts. Participants: We enrolled ambulatory adults with a positive COVID-19 test and/or a prescription for nirmatrelvir-ritonavir. Exposures: Receipt of 5 days of nirmatrelvir-ritonavir treatment versus no COVID-19 therapy. Main Outcome and Measures: The primary outcome was COVID-19 virologic rebound, defined as either (1) a positive SARS-CoV-2 viral culture following a prior negative culture or (2) two consecutive viral loads ≥4.0 log10 copies/milliliter after a prior reduction in viral load to <4.0 log10 copies/milliliter. Results: Compared with untreated individuals (n=55), those taking nirmatrelvir-ritonavir (n=72) were older, received more COVID-19 vaccinations, and were more commonly immunosuppressed. Fifteen individuals (20.8%) taking nirmatrelvir-ritonavir experienced virologic rebound versus one (1.8%) of the untreated (absolute difference 19.0% [95%CI 9.0-29.0%], P=0.001). In multivariable models, only N-R was associated with VR (AOR 10.02, 95%CI 1.13-88.74). VR occurred more commonly among those with earlier nirmatrelvir-ritonavir initiation (29.0%, 16.7% and 0% when initiated days 0, 1, and ≥2 after diagnosis, respectively, P=0.089). Among participants on N-R, those experiencing rebound had prolonged shedding of replication-competent virus compared to those that did not rebound (median: 14 vs 3 days). Only 8/16 with virologic rebound reported worsening symptoms (50%, 95%CI 25%-75%); 2 were completely asymptomatic. We detected no post-rebound nirmatrelvir-resistance mutations in the NSP5 protease gene. Conclusions and Relevance: Virologic rebound occurred in approximately one in five people taking nirmatrelvir-ritonavir and often occurred without worsening symptoms. Because it is associated with replication-competent viral shedding, close monitoring and potential isolation of those who rebound should be considered.

Characterization of Virologic Rebound Following Nirmatrelvir-Ritonavir Treatment for Coronavirus Disease 2019 (COVID-19).

We enrolled 7 individuals with recurrent symptoms or antigen test conversion following nirmatrelvir-ritonavir treatment. High viral loads (median 6.1 log10 copies/mL) were detected after rebound for a median of 17 days after initial diagnosis. Three had culturable virus for up to 16 days after initial diagnosis. No known resistance-associated mutations were identified.

Enhancing antibody responses by multivalent antigen display on thymus-independent DNA origami scaffolds.

Multivalent antigen display is a well-established principle to enhance humoral immunity. Protein-based virus-like particles (VLPs) are commonly used to spatially organize antigens. However, protein-based VLPs are limited in their ability to control valency on fixed scaffold geometries and are thymus-dependent antigens that elicit neutralizing B cell memory themselves, which can distract immune responses. Here, we investigated DNA origami as an alternative material for multivalent antigen display in vivo, applied to the receptor binding domain (RBD) of SARS-CoV2 that is the primary antigenic target of neutralizing antibody responses. Icosahedral DNA-VLPs elicited neutralizing antibodies to SARS-CoV-2 in a valency-dependent manner following sequential immunization in mice, quantified by pseudo- and live-virus neutralization assays. Further, induction of B cell memory against the RBD required T cell help, but the immune sera did not contain boosted, class-switched antibodies against the DNA scaffold. This contrasted with protein-based VLP display of the RBD that elicited B cell memory against both the target antigen and the scaffold. Thus, DNA-based VLPs enhance target antigen immunogenicity without generating off-target, scaffold-directed immune memory, thereby offering a potentially important alternative material for particulate vaccine design.

Vaccine breakthrough infection leads to distinct profiles of neutralizing antibody responses by SARS-CoV-2 variant.

Protective immunity against SARS-CoV-2 infection after COVID-19 vaccination may differ by variant. We enrolled vaccinated (n = 39) and unvaccinated (n = 11) individuals with acute, symptomatic SARS-CoV-2 Delta or Omicron infection and performed SARS-CoV-2 viral load quantification, whole-genome sequencing, and variant-specific antibody characterization at the time of acute illness and convalescence. Viral load at the time of infection was inversely correlated with antibody binding and neutralizing antibody responses. Across all variants tested, convalescent neutralization titers in unvaccinated individuals were markedly lower than in vaccinated individuals. Increases in antibody titers and neutralizing activity occurred at convalescence in a variant-specific manner. For example, among individuals infected with the Delta variant, neutralizing antibody responses were weakest against BA.2, whereas infection with Omicron BA.1 variant generated a broader response against all tested variants, including BA.2.

Viral Kinetics of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Omicron Infection in mRNA-Vaccinated Individuals Treated and Not Treated with Nirmatrelvir-Ritonavir.

We measured viral kinetics of SARS-CoV-2 Omicron infection in 36 mRNA-vaccinated individuals, 11 of whom were treated with nirmatrelvir-ritonavir (NMV-r). We found that NMV-r was associated with greater incidence of viral rebound compared to no treatment. For those that did not rebound, NMV-r significantly reduced time to PCR conversion.

Monoclonal antibody treatment drives rapid culture conversion in SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monoclonal antibodies (mAbs) are among the treatments recommended for high-risk ambulatory persons with coronavirus 2019 (COVID-19). Here, we study viral culture dynamics post-treatment in a subset of participants receiving the mAb bamlanivimab in the ACTIV-2 trial (ClinicalTrials.gov: NCT04518410). Viral load by qPCR and viral culture are performed from anterior nasal swabs collected on study days 0 (day of treatment), 1, 2, 3, and 7. Treatment with mAbs results in rapid clearance of culturable virus. One day after treatment, 0 of 28 (0%) participants receiving mAbs and 16 of 39 (41%) receiving placebo still have culturable virus (p < 0.0001). Recrudescence of culturable virus is detected in three participants with emerging mAb resistance and viral RNA rebound. While further studies are necessary to fully define the relationship between shed culturable virus and transmission, these results raise the possibility that mAbs may offer immediate (household) and public-health benefits by reducing onward transmission.