Comparisons of Pediatric and Adult SARS-CoV-2-Specific Antibodies up to 6 Months after Infection, Vaccination, or Hybrid Immunity.

BACKGROUND: Characterization of longitudinal SARS-CoV-2-specific antibody responses in children following infection and vaccination is needed to inform SARS-CoV-2 vaccine policy decisions for children, which may differ from adults. METHODS: We enrolled individuals at the time of SARS-CoV-2 infection or vaccination for longitudinal serological testing and compared SARS-CoV-2-spike-specific IgG and neutralization activity in children and adults stratified by infection and vaccination status using enzyme-linked immunosorbent and virus neutralization assays. RESULTS: Between June 2020 and December 2022, we collected sera from 669 participants aged 40 days to 55 years, including 330 unvaccinated individuals with laboratory-confirmed SARS-CoV-2 infection, 180 vaccinated SARS-CoV-2-naïve individuals, and 159 vaccinated previously infected individuals. Half (n = 330, 49.3%) were children. SARS-CoV-2-specific IgG and neutralization activity in children < 12 years old in response to infection persisted at higher levels than those of adults through at least 6 months (spike-specific IgG levels, 2.05 [95% CI: 1.4-3.1] times higher than adults; neutralizing activity, median 88.8 vs 75.2%, respectively, p = .04). In addition, all pediatric participants had significantly higher IgG levels compared with adults at 6 months following infection or vaccination, regardless of prior infection status. Vaccine-induced SARS-CoV-2-specific IgG responses in previously infected individuals persisted at higher levels than those from infection alone at 6 months (median AUC, children 5-11 years old, 9115 vs 368; adolescents 3613 vs 475; adults 1956 vs 263, all p < .001). CONCLUSIONS: These data demonstrate the robust and persistent immunologic response of SARS-CoV-2 vaccination in children and emphasize the benefit of vaccination after SARS-CoV-2 infection.

Effect of wash media type during PBMC isolation on downstream characterization of SARS-CoV-2-specific T cells.

Protocols for the isolation of peripheral blood mononuclear cells (PBMCs) from whole blood vary greatly between laboratories, especially in published studies of SARS-CoV-2-specific T cell responses following infection and vaccination. Research on the effects of different wash media types or centrifugation speeds and brake usage during the PBMC isolation process on downstream T cell activation and functionality is limited. Blood samples from 26 COVID-19-vaccinated participants were processed with different PBMC isolation methods using either PBS or RPMI as the wash media with high centrifugation speed and brakes or RPMI as the wash media with low speed and brakes (RPMI+ method). SARS-CoV-2 spike-specific T cells were quantified and characterized via a flow cytometry-based activation induced markers (AIM) assay and an interferon-γ (IFNγ) FluoroSpot assay and responses were compared between processing methods. Samples washed with RPMI showed higher AIM+ CD4 T cell responses than those washed with PBS and showed a shift away from naïve and towards an effector memory phenotype. The activation marker OX40 showed higher SARS-CoV-2 spike-induced upregulation on RPMI-washed CD4 T cells, while differences in CD137 upregulation were minimal between processing methods. The magnitude of the AIM+ CD8 T cell response was similar between processing methods but showed higher stimulation indices. Background frequencies of CD69+ CD8 T cells were increased in PBS-washed samples and were associated with higher baseline numbers of IFNγ-producing cells in the FluoroSpot assay. Slower braking in the RPMI+ method did not improve detection of SARS-CoV-2-specific T cells and caused longer processing times. Thus, the use of RPMI media with full centrifugation brakes during the wash steps of PBMC isolation was found to be most effective and efficient. Further studies are needed to elucidate the pathways involved in RPMI-mediated preservation of downstream T cell activity.

Twelve-Month Longitudinal Serology in SARS-CoV-2 Naïve and Experienced Vaccine Recipients and Unvaccinated COVID-19-Infected Individuals.

Longitudinal data comparing SARS-CoV-2 serology in individuals following infection and vaccination over 12 months are limited. This study compared the magnitude, decay, and variability in serum IgG, IgA, and neutralizing activity induced by natural infection (n = 218) or mRNA vaccination in SARS-CoV-2 naïve (n = 143) or experienced (n = 122) individuals over time using enzyme-linked immunosorbent assays and an in vitro virus neutralization assay. Serological responses were found to be highly variable after natural infection compared with vaccination but durable through 12 months. Antibody levels in vaccinated, SARS-CoV-2 naïve individuals peaked by 1 month then declined through 9 months, culminating in non-detectable SARS-CoV-2-specific serum IgA. Individuals with both infection and vaccination showed SARS-CoV-2-specific IgG and IgA levels that were more robust and slower to decline than the other groups; neutralizing activity remained highest in this group at 9 months past vaccination. These data reinforce the benefit of vaccination after SARS-CoV-2 recovery.

Human Milk SARS-CoV-2 Antibodies up to 6 Months After Vaccination.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies have been detected in human milk up to 6 weeks post-coronavirus disease 2019 (COVID-19) vaccination. We evaluated SARS-CoV-2-specific antibodies, neutralization activity, effect of pasteurization, and persistence through 6 months after vaccination. METHODS: This prospective longitudinal study enrolled 30 pregnant or lactating women. SARS-CoV-2 antibodies and neutralization capacity were analyzed using an enzyme-linked immunosorbent assay compared at prevaccination and 1, 3, and 6 months postvaccination, and through Holder pasteurization. RESULTS: Human milk SARS-CoV-2-specific IgG levels peaked at 1 month postvaccination and persisted above prevaccination levels for at least 6 months (P = .005). SARS-CoV-2-specific IgA was detected at 1 and 3 months (both P < .001) but waned by 6 months compared with baseline (P = .07). Milk SARS-CoV-2-specific IgG and IgA correlated with serum IgG at the same time point (R2 = 0.37, P < .001 and R2 = 0.19, P < .001). Neutralization activity was seen in 83.3%, 70.4%, and 25.0% of milk samples at 1, 3, and 6 months postvaccination. Neutralization most strongly correlated with SARS-CoV-2-specific IgG (R2 = 0.57, P < .001). Pre- and postpasteurization samples showed similar IgG (0.84 vs 1.07, P = .36) and neutralizing activity (57.7% vs 58.7% inhibition, P = .27), but lower IgM and IgA levels postpasteurization (0.09 vs 0.06, P = .004 and 0.21 vs 0.18, P = .043). CONCLUSIONS: The data suggest that human milk SARS-CoV-2-specific antibodies may be available to milk-fed infants for up to 6 months. In addition, donor milk from vaccinated mothers retain IgG and neutralizing activity.

Clinical manifestations of COVID-19 differ by age and obesity status.

BACKGROUND: Age and obesity status are associated with severe outcomes among hospitalized individuals with COVID-19. It remains unclear whether age and obesity are risk factors for milder COVID-19 illness. METHODS: We prospectively enrolled SARS-CoV-2-exposed individuals. Participants recorded symptoms for 28 days and were tested for SARS-CoV-2 by reverse transcription polymerase chain reaction (RT-PCR) and serology. Type, number, and duration of symptoms and SARS-CoV-2 laboratory parameters were compared by age and obesity status. RESULTS: Of 552 individuals enrolled from June 2020 to January 2021, 470 (85.1%) tested positive for SARS-CoV-2 including 261 (55.5%) adults ≥18 years, 61 (13.0%) adolescents 12-17 years, and 148 (31.5%) children <12 years. Children had fewer symptoms (median 2 vs. 3, p < 0.001) lasting fewer days (median 5 vs. 7, p < 0.001) compared with adolescents/adults. Body mass index of 300 (63.8%) individuals classified with overweight or obesity (OWOB). Individuals with OWOB suffered more symptoms compared with individuals without OWOB (median 3 vs. 2, p = 0.037), including more cough and shortness of breath (p = 0.023 and 0.026, respectively). Adolescents with OWOB were more likely to be symptomatic (66.7% vs. 34.2%, p = 0.008) and have longer respiratory symptoms (median 7 vs. 4 days, p = 0.049) compared with adolescents without OWOB. Lower RT-PCR Ct values were found in children and symptomatic individuals compared with adolescent and adults and asymptomatic individuals, respectively (p = 0.001 and 0.022). CONCLUSIONS: Adolescents and adults with OWOB experience more respiratory symptoms from COVID-19 despite similar viral loads. These findings underscore the importance of vaccinating individuals with OWOB.

The CD28-Transmembrane Domain Mediates Chimeric Antigen Receptor Heterodimerization With CD28.

Anti-CD19 chimeric antigen receptor (CD19-CAR)-engineered T cells are approved therapeutics for malignancies. The impact of the hinge domain (HD) and the transmembrane domain (TMD) between the extracellular antigen-targeting CARs and the intracellular signaling modalities of CARs has not been systemically studied. In this study, a series of 19-CARs differing only by their HD (CD8, CD28, or IgG4) and TMD (CD8 or CD28) was generated. CARs containing a CD28-TMD, but not a CD8-TMD, formed heterodimers with the endogenous CD28 in human T cells, as shown by co-immunoprecipitation and CAR-dependent proliferation of anti-CD28 stimulation. This dimerization was dependent on polar amino acids in the CD28-TMD and was more efficient with CARs containing CD28 or CD8 HD than IgG4-HD. The CD28-CAR heterodimers did not respond to CD80 and CD86 stimulation but had a significantly reduced CD28 cell-surface expression. These data unveiled a fundamental difference between CD28-TMD and CD8-TMD and indicated that CD28-TMD can modulate CAR T-cell activities by engaging endogenous partners.