Distinguishing features of long COVID identified through immune profiling.

Post-acute infection syndromes may develop after acute viral disease1. Infection with SARS-CoV-2 can result in the development of a post-acute infection syndrome known as long COVID. Individuals with long COVID frequently report unremitting fatigue, post-exertional malaise, and a variety of cognitive and autonomic dysfunctions2-4. However, the biological processes that are associated with the development and persistence of these symptoms are unclear. Here 275 individuals with or without long COVID were enrolled in a cross-sectional study that included multidimensional immune phenotyping and unbiased machine learning methods to identify biological features associated with long COVID. Marked differences were noted in circulating myeloid and lymphocyte populations relative to the matched controls, as well as evidence of exaggerated humoral responses directed against SARS-CoV-2 among participants with long COVID. Furthermore, higher antibody responses directed against non-SARS-CoV-2 viral pathogens were observed among individuals with long COVID, particularly Epstein-Barr virus. Levels of soluble immune mediators and hormones varied among groups, with cortisol levels being lower among participants with long COVID. Integration of immune phenotyping data into unbiased machine learning models identified the key features that are most strongly associated with long COVID status. Collectively, these findings may help to guide future studies into the pathobiology of long COVID and help with developing relevant biomarkers.

The Species-Specific 282 Residue in the PB2 Subunit of the Polymerase Regulates RNA Synthesis and Replication of Influenza A Viruses Infecting Bat and Nonbat Hosts.

Bat influenza viruses are genetically distant from classical influenza A viruses (IAVs) and show distinct functional differences in their surface antigens. Nevertheless, any comparative analyses between bat and classical IAV RNA polymerases or their specific subunits are yet to be performed. In this work, we have identified signature residues present in the bat influenza virus polymerase which are responsible for its altered fitness in comparison to the classical IAVs. Through comparative sequence and structural analysis, we have identified specific positions in the PB2 subunit of the polymerase, with differential amino acid preferences among bat and nonbat IAVs. Functional screening helped us to focus upon the previously uncharacterized PB2-282 residue, which is serine in bat virus but harbors highly conserved glutamic acid in classical IAVs. Introduction of E282S mutation in the human-adapted PB2 (influenza A/H1N1/WSN/1933) drastically reduces polymerase activity and replication efficiency of the virus in human, bat, and canine cells. Interestingly, this newly identified PB2-282 residue within an evolutionary conserved "S-E-S" motif, present across different genera of influenza viruses and serving as a key regulator of RNA synthesis activity of the polymerase. In contrast, bat influenza viruses harbor an atypical "S-S-T" motif at the same position of PB2, alteration of which with the human-like "S-E-T" motif significantly enhances its (H17N10/Guatemala/164/2009) polymerase activity in human cells. Together, our data indicate that the PB2-S282 residue may serve as an inherent restriction element of the bat virus polymerase, limiting its activity in other host species. IMPORTANCE Influenza A viruses are known for their ability to perform cross-species transmission, facilitated by amino acid alterations either in the surface antigen hemagglutinin (HA) or in the polymerase subunit PB2. Recent isolation of influenza A-like viruses from bats raised concern about their epizootic and zoonotic potential. Here, we identify a novel species-specific signature present within the influenza virus polymerase that may serve as a key factor in adaptation of influenza viruses from bat to nonbat host species. The PB2-282 residue, which harbors a highly conserved glutamic acid for influenza viruses across all genera (A, B, C, and D), encompasses an atypical serine in the case of bat influenza viruses. Our data show that the human-adapted polymerase, harboring a bat-specific signature (PB2-S282,) performs poorly, while bat PB2 protein, harboring a human-specific signature (PB2-E282), shows increased fitness in human cells.

Limits and patterns of cytomegalovirus genomic diversity in humans.

Human cytomegalovirus (HCMV) exhibits surprisingly high genomic diversity during natural infection although little is known about the limits or patterns of HCMV diversity among humans. To address this deficiency, we analyzed genomic diversity among congenitally infected infants. We show that there is an upper limit to HCMV genomic diversity in these patient samples, with ∼ 25% of the genome being devoid of polymorphisms. These low diversity regions were distributed across 26 loci that were preferentially located in DNA-processing genes. Furthermore, by developing, to our knowledge, the first genome-wide mutation and recombination rate maps for HCMV, we show that genomic diversity is positively correlated with these two rates. In contrast, median levels of viral genomic diversity did not vary between putatively single or mixed strain infections. We also provide evidence that HCMV populations isolated from vascular compartments of hosts from different continents are genetically similar and that polymorphisms in glycoproteins and regulatory proteins are enriched in these viral populations. This analysis provides the most highly detailed map of HCMV genomic diversity in human hosts to date and informs our understanding of the distribution of HCMV genomic diversity within human hosts.

Rapid intrahost evolution of human cytomegalovirus is shaped by demography and positive selection.

Populations of human cytomegalovirus (HCMV), a large DNA virus, are highly polymorphic in patient samples, which may allow for rapid evolution within human hosts. To understand HCMV evolution, longitudinally sampled genomic populations from the urine and plasma of 5 infants with symptomatic congenital HCMV infection were analyzed. Temporal and compartmental variability of viral populations were quantified using high throughput sequencing and population genetics approaches. HCMV populations were generally stable over time, with ~88% of SNPs displaying similar frequencies. However, samples collected from plasma and urine of the same patient at the same time were highly differentiated with approximately 1700 consensus sequence SNPs (1.2% of the genome) identified between compartments. This inter-compartment differentiation was comparable to the differentiation observed in unrelated hosts. Models of demography (i.e., changes in population size and structure) and positive selection were evaluated to explain the observed patterns of variation. Evidence for strong bottlenecks (>90% reduction in viral population size) was consistent among all patients. From the timing of the bottlenecks, we conclude that fetal infection occurred between 13-18 weeks gestational age in patients analyzed, while colonization of the urine compartment followed roughly 2 months later. The timing of these bottlenecks is consistent with the clinical histories of congenital HCMV infections. We next inferred that positive selection plays a small but measurable role in viral evolution within a single compartment. However, positive selection appears to be a strong and pervasive driver of evolution associated with compartmentalization, affecting ≥ 34 of the 167 open reading frames (~20%) of the genome. This work offers the most detailed map of HCMV in vivo evolution to date and provides evidence that viral populations can be stable or rapidly differentiate, depending on host environment. The application of population genetic methods to these data provides clinically useful information, such as the timing of infection and compartment colonization.

RNA and ß-hemolysin of group B Streptococcus induce interleukin-1ß (IL-1ß) by activating NLRP3 inflammasomes in mouse macrophages.

The inflammatory cytokine IL-1ß is critical for host responses against many human pathogens. Here, we define Group B Streptococcus (GBS)-mediated activation of the Nod-like receptor-P3 (NLRP3) inflammasome in macrophages. NLRP3 activation requires GBS expression of the cytolytic toxin, ß-hemolysin, lysosomal acidification, and leakage. These processes allow the interaction of GBS RNA with cytosolic NLRP3. The present study supports a model in which GBS RNA, along with lysosomal components including cathepsins, leaks out of lysosomes and interacts with NLRP3 to induce IL-1ß production.

Internal Tremors and Vibrations in Long COVID: A Cross-Sectional Study.

BACKGROUND: Internal tremors and vibrations are symptoms previously described as part of neurologic disorders but not fully described as a part of long COVID. This study compared pre-pandemic comorbidities, new-onset conditions, and long COVID symptoms between people with internal tremors and vibrations as part of their long COVID symptoms and people with long COVID but without these symptoms. METHODS: The Yale Listen to Immune, Symptom and Treatment Experiences Now (LISTEN) Study surveyed 423 adults who had long COVID between May 12, 2022 and June 1, 2023. The exposure variable was long COVID symptoms of internal tremors and vibrations. The outcome variables were demographic characteristics, pre-pandemic comorbidities, new-onset conditions, other symptoms, and quality of life. RESULTS: Among study participants with long COVID, median age was 46 years (IQR, 38-56), 74% were female, 87% were Non-Hispanic White, and 158 (37%) reported "internal tremors, or buzzing/vibration" as a long COVID symptom. The two groups reported similar pre-pandemic comorbidities, but participants with internal tremors reported worse health as measured by the Euro-QoL visual analogue scale (median: 40 points [IQR, 30-60] vs. 50 points [IQR, 35-62], P = .007) and had higher rates of new-onset mast cell disorders (11% [95% CI, 7.1-18] vs. 2.6% [1.2-5.6], P = .008) and neurologic conditions (22% [95% CI, 16-29] vs. 8.3% [5.4-12], P = .004). CONCLUSIONS: Among people with long COVID, those with internal tremors and vibrations had different conditions and symptoms and worse health status compared with others who had long COVID without these symptoms.

A causal link between autoantibodies and neurological symptoms in long COVID.

Acute SARS-CoV-2 infection triggers the generation of diverse and functional autoantibodies (AABs), even after mild cases. Persistently elevated autoantibodies have been found in some individuals with long COVID (LC). Using a >21,000 human protein array, we identified diverse AAB targets in LC patients that correlated with their symptoms. Elevated AABs to proteins in the nervous system were found in LC patients with neurocognitive and neurological symptoms. Purified Immunoglobulin G (IgG) samples from these individuals reacted with human pons tissue and were cross-reactive with mouse sciatic nerves, spinal cord, and meninges. Antibody reactivity to sciatic nerves and meninges correlated with patient-reported headache and disorientation. Passive transfer of IgG from patients to mice led to increased sensitivity and pain, mirroring patient-reported symptoms. Similarly, mice injected with IgG showed loss of balance and coordination, reflecting donor-reported dizziness. Our findings suggest that targeting AABs could benefit some LC patients.

Long COVID Characteristics and Experience: A Descriptive Study From the Yale LISTEN Research Cohort.

BACKGROUND: The experience of people with long COVID needs further amplification, especially with a comprehensive focus on symptomatology, treatments, and the impact on daily life and finances. Our intent is to describe the experience of people with long COVID symptomatology and characterize the psychological, social, and financial challenges they experience. METHODS: We collected data from individuals aged 18 and older reporting long COVID as participants in the Yale Listen to Immune, Symptom and Treatment Experiences Now study. The sample population included 441 participants surveyed between May 2022 and July 2023. We evaluated their demographic characteristics, socioeconomic and psychological status, index infection period, health status, quality of life, symptoms, treatments, prepandemic comorbidities, and new-onset conditions. RESULTS: Overall, the median age of the participants with long COVID was 46 years (interquartile range [IQR]: 38-57 years); 74% were women, 86% were non-Hispanic White, and 93% were from the United States. Participants reported a low health status measured by the Euro-QoL visual analog scale, with a median score of 49 (IQR: 32-61). Participants documented a diverse range of symptoms, with all 96 possible symptom choices being reported. Additionally, participants had tried many treatments (median number of treatments: 19, IQR: 12-28). They were also experiencing psychological distress, social isolation, and financial stress. CONCLUSIONS: Despite having tried numerous treatments, participants with long COVID continued to experience an array of health and financial challenges-findings that underscore the failure of the healthcare system to address the medical needs of people with long COVID. These insights highlight the need for crucial medical, mental health, financial, and community support services, as well as further scientific investigation to address the complex impact of long COVID.

Sex differences in symptomatology and immune profiles of Long COVID.

Strong sex differences in the frequencies and manifestations of Long COVID (LC) have been reported with females significantly more likely than males to present with LC after acute SARS-CoV-2 infection 1-7 . However, whether immunological traits underlying LC differ between sexes, and whether such differences explain the differential manifestations of LC symptomology is currently unknown. Here, we performed sex-based multi-dimensional immune-endocrine profiling of 165 individuals 8 with and without LC in an exploratory, cross-sectional study to identify key immunological traits underlying biological sex differences in LC. We found that female and male participants with LC experienced different sets of symptoms, and distinct patterns of organ system involvement, with female participants suffering from a higher symptom burden. Machine learning approaches identified differential sets of immune features that characterized LC in females and males. Males with LC had decreased frequencies of monocyte and DC populations, elevated NK cells, and plasma cytokines including IL-8 and TGF-ß-family members. Females with LC had increased frequencies of exhausted T cells, cytokine-secreting T cells, higher antibody reactivity to latent herpes viruses including EBV, HSV-2, and CMV, and lower testosterone levels than their control female counterparts. Testosterone levels were significantly associated with lower symptom burden in LC participants over sex designation. These findings suggest distinct immunological processes of LC in females and males and illuminate the crucial role of immune-endocrine dysregulation in sex-specific pathology.

The PAX LC Trial: A Decentralized, Phase 2, Randomized, Double-Blind Study of Nirmatrelvir/Ritonavir Compared with Placebo/Ritonavir for Long COVID.

BACKGROUND: Individuals with long COVID lack evidence-based treatments and have difficulty participating in traditional site-based trials. Our digital, decentralized trial investigates the efficacy and safety of nirmatrelvir/ritonavir, targeting viral persistence as a potential cause of long COVID. METHODS: The PAX LC trial (NCT05668091) is a Phase 2, 1:1 randomized, double-blind, superiority, placebo-controlled trial in 100 community-dwelling, highly symptomatic adult participants with long COVID residing in the 48 contiguous US states to determine the efficacy, safety, and tolerability of 15 days of nirmatrelvir/ritonavir compared with placebo/ritonavir. Participants are recruited via patient groups, cultural ambassadors, and social media platforms. Medical records are reviewed through a platform facilitating participant-mediated data acquisition from electronic health records nationwide. During the drug treatment, participants complete daily digital diaries using a web-based application. Blood draws for eligibility and safety assessments are conducted at or near participants' homes. The study drug is shipped directly to participants' homes. The primary endpoint is the PROMIS-29 Physical Health Summary Score difference between baseline and Day 28, evaluated by a mixed model repeated measure analysis. Secondary endpoints include PROMIS-29 (Mental Health Summary Score and all items), Modified GSQ-30 with supplemental symptoms questionnaire, COVID Core Outcome Measures for Recovery, EQ-5D-5L (Utility Score and all items), PGIS 1 and 2, PGIC 1 and 2, and healthcare utilization. The trial incorporates immunophenotyping to identify long COVID biomarkers and treatment responders. CONCLUSION: The PAX LC trial uses a novel decentralized design and a participant-centric approach to test a 15-day regimen of nirmatrelvir/ritonavir for long COVID.