Despite evidence of varying vaccine effectiveness, T cell responses to rotavirus (RV) vaccines remain incompletely studied. To address this research gap, RV-specific T cells in the blood of infants pre- and post-monovalent RV vaccination (RV1) were analyzed for memory recall and functionality using RV-specific peptide pool stimulation. We find that RV vaccine elicits heterogenous responses with respect to cellular and humoral immunity. T cell responses to RV vaccine are detectable in the periphery, though poorly functional. Vaccination induces Th2-biased conventional effector memory and central memory CD4 + T cells, as suggested by chemokine receptor profiles, though the response wanes by 8 months post vaccination. The presence of preexisting immunity results in no significant increase in either RV-specific IgA or T cells after vaccination. Our data provides the first in-depth assessment of RV-specific T cell responses induced by vaccine, demonstrating patterns of negative and positive association with response that may play a role in protection against rotavirus disease.

Abstract Background Mucosal immunity may contribute to clearing SARS-CoV-2 infection prior to systemic infection. We describe detection of SARS-CoV-2 specific nasal mucosal antibodies in a group of exposed household individuals that evaded systemic infection and remained seronegative.Figure 1.SARS-CoV-2-specific nasal SIgA. SARS-CoV-2-specific nasal SIgA in control and exposed participants (A). Positive thresholds of 0.20 and 6.30 AU/mL were calculated for unadjusted and adjusted SIgA, respectively, based on mean + 3 standard deviation of the control samples. Exposed participants were stratified by the positive threshold and unadjusted (B) SIgA and adjusted (C) SIgA were compared to controls and infected participants. Wilcoxon rank-sum tests determined if medians differed significantly. Methods Between June 2020 and February 2023, nasopharyngeal swabs (NPS) samples and acute and convalescent blood were collected from individuals exposed to a SARS-CoV-2-confirmed household member. Nasal secretory IgA (SIgA) antibodies targeting the SARS-CoV-2 spike protein were measured using a modified ELISA.Figure 2.SARS-CoV-2-specific nasal unadjusted (A) and adjusted (B) SIgA levels at Day 1 (enrollment), Day 8, and Day 15 for exposed seronegative with positive nasal SIgA and infected participants. SARS-CoV-2-specific nasal unadjusted (A) and adjusted (B) SIgA levels at Day 1 (enrollment), Day 8, and Day 15 for exposed seronegative with positive nasal SIgA and infected participants. Wilcoxon signed-rank tests were used to determine if medians (gray) differed significantly. Results Of 36 exposed individuals without SARS-CoV-2 detected by RT-PCR of NPS specimens and seronegative for SARS-CoV-2-specific IgG at acute and convalescent visits, 13 (36.1%) had positive SARS-CoV-2-specific SIgA levels detected in the nasal mucosa. These individuals had significantly higher nasal SIgA (median 0.52 AU/mL) compared with never-exposed, never-infected controls (n=29, 0.001 AU/mL) and infected-family members (n=43, 0.0002 AU/mL) during the acute visit, respectively (both P< 0.001, Figure 1). The nasal SARS-CoV-2-specific SIgA decreased rapidly over two weeks in the exposed seronegative individuals (Day 1 [0.52 AU/mL] vs. Day 8 [0.18 AU/mL] vs. Day 15 [median 0.02 AU/mL], P = 0.001 and 0.13, respectively, Figure 2). In contrast, SIgA increased in infected family members (Day 1 [0.0002 AU/mL] vs. Day 8 [0.65 AU/mL] vs. Day 15 [median 1.14 AU/mL], P < 0.001 and P = 0.09, respectively, Figure 2). Conclusion Transient nasal SARS-CoV-2-specific SIgA detected in exposed, seronegative individuals may have a protective role in preventing systemic infection. Elucidating factors associated with the induction of nasal SIgA response that prevent systemic invasion may inform infection and transmission prevention strategies. Disclosures Pia S. Pannaraj, MD, MPH, AstraZeneca: Grant/Research Support|Pfizer: Grant/Research Support

Abstract Background SARS-CoV-2-specific mucosal antibodies inhibit viral replication and infection within the upper respiratory tract. We investigated nasal secretory IgA (SIgA) induction differences by exposure history and protection against subsequent infection. Visual schematic of exposure groups. Methods Between June 2020 and February 2024, nasopharyngeal swab (NPS) samples were collected 14-89 days following recent infection or vaccination. SARS-CoV-2-specific nasal SIgA adjusted for total IgA (SIgAadj) and nasal neutralization activity were measured using ELISA and a surrogate virus neutralization test kit, respectively. Subsequent infection risk was compared in participants stratified by mucosal antibody levels using a Cox proportional hazards model. Figure 2 SARS-CoV-2 specific nasal secretory IgA adjusted for total IgA (SIgAadj) by exposure group compared using Wilcoxon rank sum tests. Results NPS were collected from participants with SARS-CoV-2 infection only (n=94), vaccination only (n=94), infection then vaccination (n=51), and breakthrough infection (n=94) (Fig 1). Vaccinated individuals received 3 doses of BNT162b2 (n=199, 82.7%) or mRNA-1273 (n=40, 17.3%). The mean age was 28.2 years (range 0.4-75.1), and 197 (59.2%) were female. Individuals with breakthrough infection after vaccination mounted the highest nasal SIgAadj response compared to those with infection-only (median 10.8 vs. 1.3 AU/mL, P < 0.001), vaccination-only (median 0.2 AU/mL, P< 0.001), or prior infection then vaccinated (median 3.2 AU/mL, P = 0.016) (Fig 2). Neutralization activity correlated with nasal SIgAadj (ρ = 0.48, P < 0.001). If vaccination was the most recent exposure, SIgA did not correlate with serum IgG (ρ = 0.08, P = 0.3). Forty new SARS-CoV-2 infections occurred during the 12-month follow-up period (Fig 3). In a multivariable analysis to account for age, gender, and serum IgG, participants with the lowest quartile of nasal SIgAadj levels had 5.8 times higher odds of infection than those with the higher three quartiles of nasal SIgAadj levels within 3 months (P = 0.005) (Table 1). Figure 3 Kaplan-Meier survival curves of time to subsequent SARS-CoV-2 infection in participants stratified by SARS-CoV-2-specific nasal adjusted SIgA upper 75% quantiles (high) vs. lower 25% quartile (low) over 12 months. P = 0.004 for 0-3 months and P = 0.029 for 0-12 months. Conclusion Our data demonstrate the protective role of nasal SIgA in lowering SARS-CoV-2 infection risk. Hybrid immunity induced the most robust mucosal response, suggesting a mucosal vaccine boost following intramuscular priming may help maximize mucosal immunity to prevent infection and community transmission. Predictors of subsequent infection within 0-3, 3-6, and 6-12 months using Cox proportional hazards regression model. Disclosures Pia S. Pannaraj, MD, MPH, AstraZeneca: Grant/Research Support|Pfizer: Grant/Research Support

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-naive 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.

Background: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants demonstrate predilection for different regions of the respiratory tract. While saliva-based reverse transcription-polymerase chain reaction (RT-PCR) testing is a convenient, cost-effective alternative to nasopharyngeal swabs (NPS), few studies to date have investigated whether saliva sensitivity differs across variants of concern. Methods: SARS-CoV-2 RT-PCR was performed on paired NPS and saliva specimens collected from individuals with acute coronavirus disease 2019 (COVID-19) symptoms or exposure to a COVID-19 household contact. Viral genome sequencing of NPS specimens and Los Angeles County surveillance data were used to determine the variant of infection. Saliva sensitivity was calculated using NPS-positive RT-PCR as the reference standard. Factors contributing to the likelihood of saliva SARS-CoV-2 RT-PCR positivity were evaluated with univariate and multivariable analyses. Results: Between June 2020 and December 2022, 548 saliva samples paired with SARS-CoV-2 positive NPS samples were tested by RT-PCR. Overall, saliva sensitivity for SARS-CoV-2 detection was 61.7% (95% CI, 57.6%-65.7%). Sensitivity was highest with Delta infection (79.6%) compared to pre-Delta (58.5%) and Omicron (61.5%) (P = 0.003 and 0.01, respectively). Saliva sensitivity was higher in symptomatic individuals across all variants compared to asymptomatic cases [pre-Delta 80.6% vs 48.3% (P < 0.001), Delta 100% vs 72.5% (P = 0.03), Omicron 78.7% vs 51.2% (P < 0.001)]. Infection with Delta, symptoms, and high NPS viral load were independently associated with 2.99-, 3.45-, and 4.0-fold higher odds of SARS-CoV-2 detection by saliva-based RT-PCR (P = 0.004, <0.001, and <0.001), respectively. Conclusions: As new variants emerge, evaluating saliva-based testing approaches may be crucial to ensure effective virus detection.

Mucosal immunity may contribute to clearing SARS-CoV-2 infection prior to systemic infection, thereby allowing hosts to remain seronegative. We describe the meaningful detection of SARS-CoV-2-specific nasal mucosal antibodies in a group of exposed-household individuals that evaded systemic infection. Between June 2020 and February 2023, nasopharyngeal swab (NPS) and acute and convalescent blood were collected from individuals exposed to a SARS-CoV-2-confirmed household member. Nasal secretory IgA (SIgA) antibodies targeting the SARS-CoV-2 spike protein were measured using a modified ELISA. Of the 36 exposed individuals without SARS-CoV-2 detected by the RT-PCR of NPS specimens and seronegative for SARS-CoV-2-specific IgG at enrollment and convalescence, 13 (36.1%) had positive SARS-CoV-2-specific SIgA levels detected in the nasal mucosa at enrollment. These individuals had significantly higher nasal SIgA (median 0.52 AU/mL) compared with never-exposed, never-infected controls (0.001 AU/mL) and infected-family participants (0.0002 AU/mL) during the acute visit, respectively (both p < 0.001). The nasal SARS-CoV-2-specific SIgA decreased rapidly over two weeks in the exposed seronegative individuals compared to a rise in SIgA in infected-family members. The nasal SARS-CoV-2-specific SIgA may have a protective role in preventing systemic infection.

Pregnancy poses a greater risk for severe COVID-19; however, underlying immunological changes associated with SARS-CoV-2 during pregnancy are poorly understood. We defined immune responses to SARS-CoV-2 in unvaccinated pregnant and nonpregnant women with acute and convalescent COVID-19, quantifying 217 immunological parameters. Humoral responses to SARS-CoV-2 were similar in pregnant and nonpregnant women, although our systems serology approach revealed distinct antibody and FcγR profiles between pregnant and nonpregnant women. Cellular analyses demonstrated marked differences in NK cell and unconventional T cell activation dynamics in pregnant women. Healthy pregnant women displayed preactivated NK cells and γδ T cells when compared with healthy nonpregnant women, which remained unchanged during acute and convalescent COVID-19. Conversely, nonpregnant women had prototypical activation of NK and γδ T cells. Activation of CD4+ and CD8+ T cells and T follicular helper cells was similar in SARS-CoV-2–infected pregnant and nonpregnant women, while antibody-secreting B cells were increased in pregnant women during acute COVID-19. Elevated levels of IL-8, IL-10, and IL-18 were found in pregnant women in their healthy state, and these cytokine levels remained elevated during acute and convalescent COVID-19. Collectively, we demonstrate perturbations in NK cell and γδ T cell activation in unvaccinated pregnant women with COVID-19, which may impact disease progression and severity during pregnancy.

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.

As the establishment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell memory in children remains largely unexplored, we recruited convalescent COVID-19 children and adults to define their circulating memory SARS-CoV-2-specific CD4+ and CD8+ T cells prior to vaccination. We analyzed epitope-specific T cells directly ex vivo using seven HLA class I and class II tetramers presenting SARS-CoV-2 epitopes, together with Spike-specific B cells. Unvaccinated children who seroconverted had comparable Spike-specific but lower ORF1a- and N-specific memory T cell responses compared with adults. This agreed with our TCR sequencing data showing reduced clonal expansion in children. A strong stem cell memory phenotype and common T cell receptor motifs were detected within tetramer-specific T cells in seroconverted children. Conversely, children who did not seroconvert had tetramer-specific T cells of predominantly naive phenotypes and diverse TCRαβ repertoires. Our study demonstrates the generation of SARS-CoV-2-specific T cell memory with common TCRαβ motifs in unvaccinated seroconverted children after their first virus encounter.

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.

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.

Objectives: Studies of household transmission of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) focused on households with children are limited. We investigated household secondary attack rate (SAR), transmission dynamics, and contributing factors in households with children.Materials and Methods: In this prospective case-ascertained study in Los Angeles County, California, all households members were enrolled if ≥1 member tested positive for SARS-CoV-2 by polymerase chain reaction (PCR). Nasopharyngeal PCRs, serology, and symptom data were obtained over multiple visits.Results: A total of 489 individuals in 105 households were enrolled from June to December 2020. The majority (77.3%) reported a household annual income of <$50,000, and most (92.9%) were of Hispanic/Latinx ethnicity. Children <18 years old accounted for 46.9% index cases, of whom 45.3% were asymptomatic. Household index cases were predominantly children during low community transmission and adults during the high community transmission period (χ2 = 7.647, p = 0.0036. The mean household SAR was 77.0% (95% CI: 69.4–84.6%). Child and adult index cases both efficiently transmitted SARS-CoV-2 within households [81.9%, (95% CI: 72.1–91.9%) vs. 72.4% (95% CI: 59.8–85.1%), p = 0.23]. Household income and pets were significantly associated with higher SAR in the multivariable analysis of household factors (p = 0.0013 and 0.004, respectively).Conclusions: The SAR in households with children in an urban setting with a large ethnic minority population is much higher than previously described. Children play important roles as index cases. SAR was disproportionately impacted by household income. Vaccination and public health efforts need special focus on children and vulnerable communities to help mitigate SARS-CoV-2 spread.

Background: Household transmission of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) significantly contributes to increased community cases. Methods: We conducted a prospective case-ascertained study to investigate the household secondary attack rate (SAR) and contributing factors in urban Los Angeles, California, USA. Household members were enrolled prospectively if ≥1 member tested positive for SARS-CoV-2 by polymerase chain reaction (PCR). Nasopharyngeal swabs for PCR and symptom data were obtained over multiple longitudinal visits. Serology was obtained at enrollment. Findings: A total of 489 individuals in 105 households were enrolled from June to December 2020. The mean household SAR was 77.7% (95%CI: 71.4-84.0). A third (32.1%) of index cases were asymptomatic. High SAR was associated with both symptomatic and asymptomatic index cases (76.4% [95%CI: 68.8-83.9%] vs. 79.7% [95% CI: 66.2-93.3%], p=0.66). The most common age for index cases was children <18 years old during low community transmission periods and adults ≥18 years old during peak community transmission (p=0.003). Children and adults index cases both efficiently transmitted SARS-CoV-2 within households (SAR 74.0% [95%CI: 63.4-84.6%] vs. 81.3% [95%CI: 72.3-90.3%], p=0.49). Hispanic/Latinx ethnicity was significantly associated with higher SAR in the multivariable analysis of household factors (p= 0.030). Interpretation: The SAR in our urban setting with large ethnic minority populations is much higher than previously described. SAR was disproportionately and significantly impacted by Hispanic/Latinx ethnicity. Asymptomatic individuals and children play important roles as index cases. Future vaccination and public health efforts need special focus on these groups to help mitigate SARS-CoV-2 spread.Funding: NIH/NIAID U01AI144616-02S1, Open PhilanthropyDeclaration of Interests: PSP has received consultant fees from Sanofi-Pasteur and Seqirus. She also receives research funding from AstraZeneca for an unrelated study. AG has received consultant fees from Janssen. All other authors have no conflicts of interest to report.Ethics Approval Statement: The study was approved by the Institutional Review Board at Children'sHospital Los Angeles.

Testing efforts for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been burdened by the scarcity of testing materials and personal protective equipment for health care workers. The simple and painless process of saliva collection allows for widespread testing, but enthusiasm is hampered by variable performance compared to that of nasopharyngeal swab (NPS) samples. We prospectively collected paired NPS and saliva samples from a total of 300 unique adult and pediatric patients. SARS-CoV-2 RNA was detected in 32.2% (97/300) of the individuals using the TaqPath COVID-19 Combo kit (Thermo Fisher). Performance of saliva and NPS was compared against the total number of positives regardless of specimen type. The overall concordances for saliva and NPS were 91.0% (273/300) and 94.7% (284/300), respectively. The values for positive percent agreement (PPA) for saliva and NPS were 81.4% (79/97) and 89.7% (87/97), respectively. Saliva yielded detection of 10 positive cases that were negative by NPS. For symptomatic and asymptomatic pediatric patients not previously diagnosed with COVID-19, the performances of saliva and NPS were comparable (PPA, 82.4% versus 85.3%). The overall values for PPA for adults were 83.3% and 90.7% for saliva and NPS, respectively, with saliva yielding detection of 4 fewer cases than NPS. However, saliva performance for symptomatic adults was identical to NPS performance (PPA of 93.8%). With lower cost and self-collection capabilities, saliva can be an appropriate sample choice alternative to NPS for detection of SARS-CoV-2 in children and adults.