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BackgroundOlder adults have been disproportionately affected during the SARS-CoV-2 pandemic, including higher risk of severe disease and long-COVID. Prior exposure to endemic human coronaviruses (HCoV) may modulate the response to SARS-CoV-2 infection and contribute to age-related observations. We hypothesized that cross-reactive antibodies to SARS-CoV-2 are associated with antibodies to HCoV and that both increase with age.MethodsTo assess SARS-CoV-2 unexposed individuals, we drew upon archived anonymized residual sero-surveys conducted in British Columbia (BC), Canada, including before SARS-CoV-2 emergence (May, 2013) and before widespread community circulation in BC (May, 2020). Fifty sera, sex-balanced per ten-year age band, were sought among individuals ≤10 to ≥80 years old, supplemented as indicated by sera from March and September 2020. Sera were tested on the Meso Scale Diagnostics (MSD) electrochemiluminescent multiplex immunoassay to quantify IgG antibody against the Spike proteins of HCoV, including alpha (HCoV-229E, HCoV-NL63) and beta (HCoV-HKU1, HCoV-OC43) viruses, and the 2003 epidemic beta coronavirus, SARS-CoV-1. Cross-reactive antibodies to Spike, Nucleocapsid, and the Receptor Binding Domain (RBD) of SARS-CoV-2 were similarly measured, with SARS-CoV-2 sero-positivity overall defined by positivity on ≥2 targets.ResultsSamples included 407 sera from 2013, of which 17 were children ≤10 years. The 2020 samples included 488 sera, of which 88 were children ≤10 years. Anti-Spike antibodies to all four endemic HCoV were acquired by 10 years of age. There were 20/407 (5%) sera in 2013 and 8/488 (2%) in 2020 that were considered sero-positive for SARS-CoV-2 based on MSD testing. Of note, antibody to the single SARS-CoV-2 RBD target was detected in 329/407 (81%) of 2013 sera and 91/488 (19%) of 2020 sera. Among the SARS-CoV-2 overall sero-negative population, age was correlated with anti-HCoV antibody levels and these, notably 229E and HKU1, were correlated with cross-reactive anti-SARS-CoV-2 RBD titres. SARS-CoV-2 overall sero-positive individuals showed higher titres to HCoV more generally.ConclusionMost people have an HCoV priming exposure by 10 years of age and IgG levels are stable thereafter. Anti-HCoV antibodies can cross-react with SARS-CoV-2 epitopes. These immunological interactions warrant further investigation with respect to their implications for COVID-19 clinical outcomes.

Mycoplasma orale is an obligate intracellular bacterium usually found as a commensal in the human oral cavity. Symptomatic infections with this organism are rare, but severe disease has been described in the setting of impaired humoral immunity. Here, we describe a case in which M. orale was identified from the joint fluid of a patient with septic arthritis, splenic lesions, and agammaglobulinemia. A 15-year-old boy was admitted to the hospital with fever, progressive left knee swelling, and pain. His medical history was significant for Burkitt’s lymphoma, the treatment of which had included rituximab 6 years earlier. M. orale was identified in the synovial fluid using 16S ribosomal RNA gene sequencing. He was also found to be hypogammaglobulinemic, and imaging revealed multiple splenic lesions. He was treated with doxycycline and intravenous immunoglobulin, which resulted in complete resolution of his arthritis and other symptoms. Mycoplasma species should be suspected in patients with humoral immunodeficiency and compatible findings.

Preexisting cross-reactivity to SARS-CoV-2 occurs in the absence of prior viral exposure. However, this has been difficult to quantify at the population level due to a lack of reliably defined seroreactivity thresholds. Using an orthogonal antibody testing approach, we estimated that about 0.6% of nontriaged adults from the greater Vancouver, Canada, area between May 17 and June 19, 2020, showed clear evidence of a prior SARS-CoV-2 infection, after adjusting for false-positive and false-negative test results. Using a highly sensitive multiplex assay and positive/negative thresholds established in infants in whom maternal antibodies have waned, we determined that more than 90% of uninfected adults showed antibody reactivity against the spike protein, receptor-binding domain (RBD), N-terminal domain (NTD), or the nucleocapsid (N) protein from SARS-CoV-2. This seroreactivity was evenly distributed across age and sex, correlated with circulating coronaviruses' reactivity, and was partially outcompeted by soluble circulating coronaviruses' spike. Using a custom SARS-CoV-2 peptide mapping array, we found that this antibody reactivity broadly mapped to spike and to conserved nonstructural viral proteins. We conclude that most adults display preexisting antibody cross-reactivity against SARS-CoV-2, which further supports investigation of how this may impact the clinical severity of COVID-19 or SARS-CoV-2 vaccine responses.

Death from sepsis in the neonatal period remains a serious threat for millions. Within 3 days of administration, bacille Calmette-Guérin (BCG) vaccination can reduce mortality from neonatal sepsis in human newborns, but the underlying mechanism for this rapid protection is unknown. We found that BCG was also protective in a mouse model of neonatal polymicrobial sepsis, where it induced granulocyte colony-stimulating factor (G-CSF) within hours of administration. This was necessary and sufficient to drive emergency granulopoiesis (EG), resulting in a marked increase in neutrophils. This increase in neutrophils was directly and quantitatively responsible for protection from sepsis. Rapid induction of EG after BCG administration also occurred in three independent cohorts of human neonates.

BackgroundQuantifying antibody reactivity against multiple SARS-CoV-2 antigens at the population level may help understand individual differences in COVID-19 severity. Pre-existing low antibody cross-reactivity may be particularly prevalent among childcare providers, including pediatric health care workers (HCW) who may be more exposed to circulating coronaviruses.MethodsCross-sectional study that included adults in the Vancouver area in British Columbia (BC), Canada, between May 17 and June 19, 2020. SARS-CoV-2 seroprevalence was ascertained by measuring total SARS-CoV-2 IgG/M/A antibodies against a recombinant spike (S1) protein and adjusted for bias due to false-positive and false-negative test results. A novel, high sensitivity multiplex assay was also used to profile IgG against four SARS-CoV-2 antigens, SARS-CoV and four circulating coronaviruses.FindingsAmong 276 participants (71% HCW), three showed evidence of direct viral exposure, yielding an adjusted seroprevalence of 0.60% [95%CI 0% – 2.71%], with no difference between HCW and non-HCW, or between paediatric and adult HCW. Among the 273 unexposed individuals, 7.3% [95%CI 4.5% – 11.1%], 48.7 [95%CI 42.7% – 54.8%] and 82.4% [95%CI 77.4% – 86.7%] showed antibody reactivity against SARS-CoV-2 RBD, N or Spike proteins, respectively. SARS-CoV-2 reactivity did not significantly correlate with age, sex, did not significantly differ between HCW and non-HCW (prevalence 1.0% vs 1.0%; P=1.00) and between pediatric and adult HCW (0.7% vs 1.6%; P=0.54), and modestly correlated with reactivity to circulating coronaviruses (Spearman rho range: 0.130 to 0.224 for 7 significant (FDR 5%), out of 16 correlations, from 36 correlations tested).InterpretationA substantial proportion of individuals showed low, but detectable antibody reactivity against SARS-CoV-2 antigens in this population despite low evidence of direct SARS-CoV-2 exposure.FundingNIAID/NIH

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult mouse models to neonatal diseases is limited. To better understand disease progression, host responses and long-term impact of interventions in neonates, a neonatal mouse model likely is a better fit. The sparse use of neonatal mouse models can in part be attributed to the technical difficulties of working with these small animals. A neonatal mouse model was developed to determine the effects of probiotic administration in early life and to specifically assess the ability to establish colonization in the newborn mouse intestinal tract. Specifically, to assess probiotic colonization in the neonatal mouse, Lactobacillus plantarum (LP) was delivered directly into the neonatal mouse gastrointestinal tract. To this end, LP was administered to mice by feeding through intra-esophageal (IE) gavage. A highly reproducible method was developed to standardize the process of IE gavage that allows an accurate administration of probiotic dosages while minimizing trauma, an aspect particularly important given the fragility of newborn mice. Limitations of this process include possibilities of esophageal irritation or damage and aspiration if gavaged incorrectly. This approach represents an improvement on current practices because IE gavage into the distal esophagus reduces the chances of aspiration. Following gavage, the colonization profile of the probiotic was traced using quantitative polymerase chain reaction (qPCR) of the extracted intestinal DNA with LP specific primers. Different litter settings and cage management techniques were used to assess the potential for colonization-spread. The protocol details the intricacies of IE neonatal mouse gavage and subsequent colonization quantification with LP.

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.