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martes, 25 de enero de 2022

La vacunación antes o después de la infección por el SARS-CoV-2 produce una respuesta....

 "Se ha demostrado que la infección natural por sí sola proporciona una protección de corta duración contra la infección, lo que demuestra la importancia de la vacunación, independientemente de los antecedentes de infección. [...] Es más seguro vacunarse antes que después de la infección".

La vacunación antes o después de la infección por el SARS-CoV-2 produce una respuesta humoral robusta y anticuerpos que neutralizan eficazmente las variantes 

Timothy A. Bates https://orcid.org/0000-0002-2533-7668Savannah K. McBride https://orcid.org/0000-0001-8585-8038Hans C. Leier https://orcid.org/0000-0002-0363-1444Gaelen Guzman https://orcid.org/0000-0002-7696-6034[...]Fikadu G. Tafesse https://orcid.org/0000-0002-8575-4164 Authors Info & Affiliations

Las vacunas actuales contra el COVID-19 reducen significativamente la morbilidad y la mortalidad general y son de vital importancia para controlar la pandemia. Los individuos que se han recuperado previamente de la COVID-19 tienen respuestas inmunitarias mejoradas después de la vacunación (inmunidad híbrida) en comparación con sus compañeros vacunados ingenuamente; sin embargo, los efectos de las infecciones de ruptura posteriores a la vacunación en la respuesta inmunitaria humoral aún están por determinar. Aquí, medimos las respuestas de anticuerpos neutralizantes de 104 individuos vacunados, incluyendo aquellos con infecciones de ruptura, inmunidad híbrida y sin historia de infección. Encontramos que los sueros inmunes humanos después de la infección por irrupción y la vacunación después de la infección natural, neutralizan ampliamente las variantes de SARS-CoV-2 en un grado similar. Mientras que la edad se correlaciona negativamente con la respuesta de anticuerpos después de la vacunación sola, no se encontró ninguna correlación con la edad en los grupos de inmunidad de ruptura o híbrida. En conjunto, nuestros datos sugieren que la exposición adicional al antígeno de la infección natural aumenta sustancialmente la cantidad, la calidad y la amplitud de la respuesta inmunitaria humoral, independientemente de que se produzca antes o después de la vacunación.


INTRODUCCIÓN


El coronavirus respiratorio agudo grave 2 (SARS-CoV-2) es el agente causante de la actual pandemia de la enfermedad por coronavirus 2019 (COVID-19). A nivel mundial, los casos siguen aumentando a pesar de las campañas de vacunación en todo el mundo. (1) Se han desarrollado numerosas vacunas seguras y efectivas que reducen eficazmente el riesgo de infección, enfermedad grave y muerte, entre ellas la BNT162b2 (Pfizer), la mRNA-1273 (Moderna) y la Ad26.COV2.S (Janssen). (2, 3) Sin embargo, las variantes preocupantes (VOC) con diferentes niveles de aumento de la transmisibilidad y la resistencia a la inmunidad existente han surgido secuencialmente, se han extendido ampliamente y han retrocedido con el tiempo desde el comienzo de la pandemia. (4-7) Varios estudios han demostrado que las respuestas de los anticuerpos de la oleada inicial de vacunas a principios de 2021 han disminuido durante los seis meses siguientes a la vacunación, lo que posiblemente ha contribuido a un aumento de las infecciones de inicio. (8-12) Las dosis de vacunas de refuerzo se aprobaron por primera vez en Israel en julio de 2021, y desde entonces se han adoptado de forma más generalizada en otros países para hacer frente a estos problemas, a pesar de la preocupación de que las campañas de refuerzo puedan desviar dosis de vacunas muy necesarias de los países con menores ingresos. (13)

Se ha informado de que la vacunación tras la recuperación de la infección natural por el SRAS-CoV-2, o "inmunidad híbrida", aumenta sustancialmente tanto la potencia como la amplitud de la respuesta humoral al SRAS-CoV-2. (14, 15) Sin embargo, los estudios actuales sobre la infección intermitente que se produce después de la vacunación se han centrado en la identificación de factores de susceptibilidad, como el título de neutralización del virus antes de la infección. (16) El impacto de la infección por irrupción en la respuesta de anticuerpos neutralizantes y su comparación con la respuesta provocada por la inmunidad híbrida sigue sin estar claro; por lo tanto, emprendimos el presente estudio para abordar directamente esta laguna de conocimiento.


RESULTADOS

Cohorte y diseño del estudio

Reclutamos un total de 104 participantes (Tabla 1) que consistían en 31 individuos totalmente vacunados con infecciones por disrupción confirmadas por PCR, 31 individuos con una (6 individuos) o dos dosis de vacuna (25 individuos) tras la recuperación de COVID-19 (inmunidad híbrida), y 42 individuos totalmente vacunados sin antecedentes de COVID-19 o infección por disrupción (Fig. 1A). Noventa y seis participantes recibieron BNT162b2, 6 recibieron ARNm-1273 y 2 recibieron Ad26.COV2.S. Se recogieron muestras de suero de cada uno de los participantes, que luego se analizaron para determinar las concentraciones de anticuerpos eficaces del 50% (EC50) mediante un ensayo inmunoabsorbente ligado a enzimas (ELISA), y el título neutralizante del 50% de SARS-CoV-2 vivo con pruebas de neutralización por reducción de foco (FRNT50) contra la cepa de linaje temprano de SARS-CoV-2 (WA1) y los aislados clínicos de tres COV: Alfa (B.1.1.7), Beta, (B.1.351), y Delta (B.1.617.2). Realizamos experimentos adicionales de fagocitosis celular dependiente de anticuerpos (ADCP) para evaluar cualquier diferencia funcional en la respuesta de anticuerpos de cada grupo


 

We first analyzed the hybrid immunity of participants who received only a single vaccine dose compared to those who had received two doses (Fig. S1). All measures of antibody levels, ADCP, and live virus neutralization revealed no significant difference between these two groups. For this reason, we combined these samples into a single group containing participants with both one and two vaccine doses following natural infection, which we henceforth refer to as the hybrid immune group.

Antibody levels following breakthrough infection, hybrid immunity, and vaccination alone

ELISA geometric mean titers (GMT) EC50 values for SARS-CoV-2 spike-specific antibodies were significantly elevated in both the breakthrough (2.5-fold, P = 0.005) and hybrid immune (3.6-fold, P < 0.0001) groups compared to vaccination alone, but we saw no significant difference between the breakthrough and hybrid groups (Fig. 1B). A similar trend was seen for EC50 values specific for the spike receptor binding domain (RBD) (Fig. 1B). We additionally confirmed that none of the vaccine-only participants exhibited reactivity against the nucleocapsid (N) protein, supporting lack of previous infection, whereas the breakthrough and hybrid immune groups were 68 and 48 percent N responsive, respectively (Fig. 1B). Opsonization with hybrid immune and breakthrough sera also induced phagocytosis of spike protein-coated particles in an ADCP assay significantly more than vaccination alone, but not compared to each other (Fig. 1C). The levels of IgG and IgA antibodies specific to RBD protein displayed a similar trend to the total EC50 levels with significant increases for hybrid immunity and breakthrough compared to vaccination alone, but not compared with each other (Fig. 1D). RBD-specific IgM values were notably low and did not differ significantly between groups. Consistent with previous reports, (17) spike-specific antibody levels correlated negatively with age among vaccine-only participants. In contrast, neither the breakthrough nor hybrid immune group recapitulated this correlation, displaying no significant age-related trend (Fig. 1E).

Neutralizing antibody titers against SARS-CoV-2 and the variants of concern

We next quantified the functional activity of participants’ immune sera by comparing their neutralization titers against early (WA1) SARS-CoV-2 and selected VOCs. Against all viruses, the trend mirrored that of the antibody EC50 levels, with the vaccine-only group FRNT50 titers significantly lower than both breakthrough and hybrid immunity, which were comparable with each other (Fig. 2A). The FRNT50 GMT of hybrid immune group participants were 10.8, 16.9, 32.8, and 15.7-fold higher than vaccination alone for WA1, Alpha, Beta, and Delta variants, while breakthrough group participants were 6.0, 11.8, 17.0, and 8.5-fold higher than vaccination alone, respectively, all with P < 0.0001. Among vaccine group participants, neutralization of the Beta variant was significantly reduced compared to WA1, while the difference seen for the hybrid immune and breakthrough groups was not significant (Fig. S2).
Fig. 2. Neutralizing antibody response following breakthrough infection, hybrid immunity, and vaccination alone.
(A) Neutralizing antibody titers determined by focus forming assay with clinical isolates of the original strain of SARS-CoV-2 (WA1), Alpha, Beta, and Delta variants. (B) The ratio of Alpha, Beta, and Delta variant neutralization to WA1 neutralization. WA1 neutralizing titer versus Alpha (C), Beta (D), and Delta (E) variant neutralizing titer. The dotted line indicates equal neutralization. Error bars in A and B indicate the geometric mean with the 95% confidence interval. P values in A were two-tailed and calculated with the Kruskal-Wallis method with Dunn’s multiple comparison correction.
In addition to eliciting immunity with greater breadth (Fig. 2A), the serum antibody potency across the breadth of VOCs tested was greater for both hybrid immune and breakthrough groups, as measured by an increase in the ratio of variant neutralization over WA1 FRNT50 values against Alpha and Beta for the hybrid immune and breakthrough groups, and against Delta for the hybrid immune group (Fig. 2B and S3). Breakthrough and hybrid immune participants grouped more tightly and displayed variant neutralizing titers closer to that of WA1 (Fig. 2C-E).

Quality of the neutralizing antibody response

We also found that hybrid immunity was associated with a remarkable improvement in the proportion of spike-specific antibodies that were also neutralizing. WA1 neutralizing titers correlated with spike-specific antibody levels for all three groups, but the hybrid immune and breakthrough groups correlated more strongly (Fig. 3A). To analyze the efficiency of sera at neutralizing a given virus strain, we determined a neutralizing potency index by calculating the ratio of neutralizing titer (FRNT50) to spike binding EC50 values. (18) The index expresses a ratio of fold-serum-dilution with 50% neutralization potency to fold-serum-dilution 50% spike binding capacity, or a relative neutralizing antibody to total antibody ratio for a given subject’s serum. The neutralizing potency index was significantly higher among hybrid immune and breakthrough participants than after vaccination alone (Fig. 3B). Lastly, we found that the relationship between age and total antibody levels also extends to neutralizing titer; vaccine-only participants displayed a clear negative correlation with age, while the hybrid immune and breakthrough participants showed no such correlation (Fig. 3C). No association was seen between reported sex and neutralizing titer for any of the groups (Fig. 3D).
Fig. 3. Neutralizing efficiency and correlation with age.
(A) Correlation between spike-specific EC50 vaules and WA1 neutralizing titers. (B) Serum neutralizing potency index was calculated as the ratio of WA1 neutralizing titer to spike-specific EC50 values. (C) Correlation between age and WA1 neutralizing titers. (D) WA1 neutralization by sex. Error bars in B and D indicate the geometric mean with the 95% confidence interval. P values in B were two-tailed and calculated with the Kruskal-Wallis method with Dunn’s multiple comparison correction. P values in D were two-tailed and calculated with using a two-way ANOVA with the Šidák multiple comparison correction. Scatter plots in A depict the simple linear fit of log transformed FRNT50 versus log transformed EC50 values with 95% confidence bands. Scatter plots in C depict the simple linear fit of log transformed FRNT50 versus age with 95% confidence bands. Correlations in A and C show spearman rank correlation coefficients and two-tailed P values.

DISCUSSION

Overall, our results show that SARS-CoV-2 infection before or after vaccination gives a significantly larger boost to the neutralizing antibody response compared to two doses of vaccine alone. More importantly, the potency and breadth of the antibody response appears to improve concomitantly. It has been well established that natural infection alone provides short-lived protection from infection, (17) showing the importance of vaccination, regardless of infection history. Because vaccination protects against severe disease and death, (19) it is safer for individuals to be vaccinated before rather than after natural infection.
The negative correlation between age and neutralizing antibody levels following vaccination alone is an effect that has been previously identified. (20) The relationship between age and antibody levels following natural infection is markedly more complex, with a peak in antibody levels seen between the ages of 60 and 80. (21) The exact reasons for this association remain to be determined, but one hypothesis is that the greater disease severity among individuals of advanced age leads to an overall greater humoral response. (18) These two opposing trends may obscure any age dependence of antibody levels in the present study among patients with humoral responses resulting from both vaccination and natural infection.
 
Estudios recientes han sugerido que la respuesta humoral continúa desarrollándose mucho tiempo después de la vacunación, con células B de memoria en puntos de tiempo tardíos después de la vacunación que muestran una mejor calidad y amplitud en comparación con los puntos de tiempo tempranos. (14, 15, 22) Nuestros datos no pueden separar la contribución de la potenciación mixta debida a la combinación de la vacunación con la infección natural, de la contribución del desarrollo de células B de memoria en curso durante el tiempo transcurrido entre la primera exposición al antígeno y la potenciación más reciente, ya sea por la vacunación o por la infección de ruptura. Futuros estudios con individuos que han sido vacunados y reforzados pueden ser capaces de distinguir entre estas posibilidades, y un primer estudio sugiere que la vacunación de refuerzo 8 meses después de una segunda dosis conduce a la mejora de los títulos neutralizantes de la variante Delta en general de 6 a 12 veces. (23) Esto parece coherente con las mejoras de 8,5 y 15,7 veces contra la variante Delta para los grupos de inmunidad de ruptura e híbrida, respectivamente, en comparación con dos dosis de vacuna solas. Esto sugiere que la magnitud de la mejora para las vacunas de refuerzo puede ser similar a las observadas con la vacunación combinada y la infección natural, incluida la inmunidad híbrida con una sola dosis de vacuna de ARNm. Esto apuntaría a la importancia del compartimento de células B de memoria en la generación de una respuesta humoral neutralizante cruzada robusta y variante. Aunque este estudio se centra en la respuesta humoral, se sabe que la respuesta celular de las células T desempeña un papel importante en la respuesta a la vacunación y a la infección por el SARS-CoV-2. (24)
Las vacunas COVID-19 que utilizan la tecnología de ARNm, incluyendo la BNT162b2 y la ARNm-1273 son las vacunas más comúnmente administradas en los Estados Unidos, donde se realizó este estudio, y la mayoría de los participantes de este estudio recibieron la vacuna BNT162b2. Sin embargo, algunos participantes recibieron la vacuna basada en el adenovirus Ad26.COV2.S. La mayoría de las investigaciones sobre la inmunidad híbrida se han centrado en la vacunación con ARNm, pero las investigaciones sobre la inmunidad híbrida de las vacunas con adenovirus han mostrado mejoras similares en los títulos de neutralización y en la neutralización cruzada de las variantes. (25) Aunque este estudio no fue diseñado para comparar la eficacia de las diferentes tecnologías de vacunación, no anticipamos ningún efecto sustancial debido a las diferencias en los tipos de vacunas.
La vacunación es muy eficaz para prevenir los resultados más graves de la COVID-19 y debe administrarse independientemente del estado de la infección previa y de la edad. Una sola dosis de la vacuna puede proporcionar suficiente protección para muchos individuos con infección previa por SARS-CoV-2. La disponibilidad de la vacuna sigue siendo limitada en muchas regiones y el camino más corto para lograr una amplia inmunidad mundial puede ser dar prioridad a la administración de al menos una dosis de vacuna al mayor número posible de personas con antecedentes confirmados de infección por el SRAS-CoV-2.

MATERIALS AND METHODS

Study design

The purpose of this study was to directly compare the humoral immune response among individuals who received COVID-19 vaccines either prior to or following naturally acquired SARS-CoV-2 infection. Serum samples were collected from participants, which were analyzed using enzyme-linked immunosorbent (ELISA) assays, focus reduction neutralization tests, and measurement of antibody dependent cellular phagocytosis. Study participants were selected for inclusion based on a history of both vaccination and previous SARS-CoV-2 infection. Vaccinated controls with no history of previous infection were selected on the basis of sex, age, days between vaccine doses, and the time period since the most recent vaccination.

Cohort selection and serum collection:

Health care workers at Oregon Health & Science University were recruited and enrolled in the study belonging to three groups: Vaccine-only, hybrid immunity, and breakthrough infection. Written informed consent was obtained at the time of enrollment and study approval was obtained from the OHSU institutional review board (IRB#00022511). Vaccine-only participants were fully vaccinated, defined as having received 2 doses of BNT162b2 or mRNA-1273, or 1 dose of Ad26.COV2.S. Serum samples were collected at least 14 days after the final vaccine dose. Hybrid immune participants had a history of PCR-confirmed diagnosis of COVID-19 at least 10 days prior to vaccination with at least one dose of BNT162b2, mRNA-1273, or Ad26.COV2.S and serum samples were collected at least 10 days after the final vaccine dose. Breakthrough participants were fully vaccinated as defined for the vaccine only group at least 10 days prior to PCR confirmed diagnosis of COVID-19 and serum samples were collected at least 10 days after the date of diagnosis. Sera were obtained by collecting 4-6 mL of whole blood in a BD Vacutainer Plus Plastic Serum Tube, which was centrifuged for 10 min at 1000xg before serum was aliquoted and stored at -20°C. Hybrid immune and breakthrough infection participants were selected based on availability while vaccine-only participants were selected to most closely match the average sex, age, and time since most recent vaccination (or infection for breakthrough) of the other two groups. Participants in these cohorts are previously described. (20, 26)

Enzyme-linked immunosorbent assays (ELISA):

ELISAs were performed as previously described. (20) In 96-well plates (Corning Incorporated, EIA/RIA High binding, Ref #359096). Plates were coated with 100 μL/well of the following proteins at 1 μg/mL in PBS and incubated overnight at 4°C with rocking: SARS-CoV-2 RBD (produced in Expi293F cells and purified using Ni-NTA chromatography), Full-length SARS-CoV-2 spike (Recombinant Spike, SARS-CoV-2 stabilized protein, produced in Expi293F cells, BEI resources #NR-52724), Nucleocapsid (SARS-CoV-2 Nucleocapsid-His, insect cell-expressed, SinoBio Cat: 40588-V08B, Item #NR-53797, lot #MF14DE1611). Plates were washed three times with 0.05% v/v Tween-20 in PBS (wash buffer) and blocked with 150 μL/well 5% nonfat dry milk powder in wash buffer (blocking buffer) at room temperature of approximately 20°C (RT) for 1 hour with rocking. Breakthrough and control sera were aliquoted and frozen in dilution plates then resuspended in blocking buffer; sera were diluted and added to ELISA plates 100 μL/well (6 × 4-fold dilutions from 1:50 to 1:51,200), except for IgM (6 × 3-fold dilutions from 1:25 to 1:6075). Sera was incubated for 1 hour at RT before plates were filled three times with wash buffer. Secondary antibodies were added to plates at 100 μL/well depending on the intended readout: Goat anti-human IGG/A/M-HRP at 1:10,000 (Invitrogen, Ref #A18847), anti-human IgA-HRP at 1:3,000 (BioLegend, Ref #411002), Mouse anti-human IgG-HRP Clone G18-145 at 1:3,000 (BD Biosciences, Ref #555788), Goat anti-human IgM-HRP at 1:3,000 (Bethyl Laboratories, Ref #A80-100P). Plates were incubated protected from light with secondary at RT for 1 hour with rocking, then filled three times with wash buffer prior to the development with o-phenylenediamine dihydrochloride (OPD, Thermo Scientific #34005) according to the manufacturer’s instructions. The reaction was stopped after 25 min using an equivalent volume of 1 M HCl; optical density was measured at 492 nm using a CLARIOstar plate reader. Normalized A492 values were calculated by subtracting the average of negative control wells and dividing by the 99th percentile of all wells from the same experiment. A dilution series of positive control serum was included on each plate to verify appropriate performance of the assay.

Cell culture:

Vero E6 monkey kidney epithelial cells (CRL-1586) were obtained from ATCC and maintained in tissue culture-treated vessels in Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), 1% nonessential amino acids (NEAA), 1% penicillin-streptomycin (PS) (complete media) in tissue culture conditions (TCC) of 100% relative humidity, 37°C, and 5% CO2. THP-1 (ATCC, TIB-202) human monocyte cells were obtained from ATCC and maintained in suspension culture in tissue culture treated vessels in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% FBS, 1% NEAA, and 1% PS (THP-1 media).

SARS-CoV-2 growth and titration:

SARS-CoV-2 isolates USA-WA1/2020 [lineage A] (NR-52281), USA/CA_CDC_5574/2020 [lineage B.1.1.7 – alpha] (NR-54011), hCoV-19/South Africa/KRISP-K005325/2020 [lineage B.1.351 – beta] (NR-54009), hCoV-19/USA/PHC658/2021 [lineage B.1.617.2 – delta] (NR-55611) were obtained from BEI Resources. Viral stocks were propagated as previously described. (5) Sub-confluent Vero E6 cells were infected at an MOI of 0.05 in a minimal volume (0.01 mL/cm2) of Opti-MEM + 2% FBS (dilution media) for 1 hour at TCC then 0.1 mL/cm2 additional complete media was added and incubated for 24 hours at TCC. Culture supernatant was centrifuged for 10 min at 1000xg and frozen at -80°C in aliquots. Titration was performed on clear 96 well tissue culture plates containing 70–90% confluent (at the time of infection) Vero E6 cells. 8 × 10-fold dilutions were prepared in dilution media and 30 μL/well of diluted virus was incubated with the cells for 1 hour at TCC before further addition of Opti-MEM, 2% FBS, 1% methylcellulose (overlay media) and incubation for 24 hours at TCC. Plates were then fixed by soaking in 4% formaldehyde in PBS for 1 hour then removing from BSL-3 following institutional biosafety protocols. Cells were permeabilized in 0.1% bovine serum albumin and 0.1% saponin in PBS (perm buffer) for 30 min, then with polyclonal anti-SARS-CoV-2 alpaca serum (Capralogics Inc.) (1:5000 in perm buffer) overnight at 4°C. Plates were washed three times with 0.01% Tween-20 in PBS (focus wash buffer), then incubated for 2 hours at RT with 1:20,000 anti-alpaca-HRP (Novus #NB7242). Plates were filled three times with focus wash buffer, then incubated with TrueBlue (Sera Care #5510-0030) for 30 min or until sufficiently developed for imaging. Well images were captures with a CTL Immunospot Analyzer and counted with Viridot (1.0) in R (3.6.3). (27) Viral stock titers in focus forming units (FFU) were calculated from the dilution factor and volume used during infection.

Focus reduction neutralization test (FRNT):

FRNT assays were carried out as described. (5) Duplicate 5x4.7-fold (1:10-1:4879) serial dilutions of participant sera were prepared in 96-well plates. An equal volume of dilution media containing approximately 50 FFU of SARS-CoV-2 or variant was added to each well (final dilutions of sera, 1:20 – 1:9760) and incubated 1 hour at TCC. Virus-serum mixtures were used to infect Vero E6 cells in 96-well plates as described above in the titration assay. Each plate contained 16 virus-only control wells, one for each serum dilution series. Fixation, development, and counting of FRNT plates was carried out as described above in the titration assay. Percent neutralization values were calculated for each well as the focus count divided by the average focus count of virus-only control wells from the same plate.

Antibody Dependent Cellular Phagocytosis (ADCP):

ADCP assay was adapted from a protocol described previously. (28) Biotinylated RBD incubated at 1μg/ml with fluorescent neutravidin beads (Invitrogen, F8775) for 2 hours at RT; beads were washed twice with 1% BSA in PBS (dilution buffer) and resuspended at a final dilution of 1:100 in dilution buffer. In a 96-well plate, 10μL of resuspended bead solution was incubated with 10μL of diluted serum from study subjects for 2 hours at 37°C. After serum pre-treatment, 2x104 THP-1 cells were added to each well in 80μL THP-1 media and incubated overnight in TCC. The following morning, 100 μL of 4% paraformaldehyde was added to each well and incubated at least 30 min at RT before analysis on a CytoFLEX flow cytometer (Beckman Coulter). Samples were mixed for 3 s prior to analysis and samples were injected until at least 2500 cell events were recorded per sample. Phagocytosis scores are reported as the product of percent bead-positive cells and mean fluorescence intensity of bead-positive cells, then divided by 106 for presentation. Three replicate experiments were performed for each participant serum sample, the average of which was used for further analysis. The gating strategy with representative data are presented in Fig. S4.

Statistical analysis:

FRNT50 and EC50 values were calculated by fitting percent neutralization or normalized A492 values to a dose-response curve as previously described. (5) Final FRNT50 values below the limit of detection (1:20) were set to 1:19. Final EC50 values below the limit of detection of 1:25 for N, Spike, RBD, IgG, IgA were set to 1:24 and values below 1:12.5 for IgM was set to 1:12. Aggregated EC50 and FRNT50 values were analyzed and plotted in Graphpad Prism (9.2.0). Dot plots of EC50 and FRNT50 values were generated on a log transformed axis with error bars showing the geometric mean and 95% confidence interval. Phagocytosis score and Neutralization ratio were plotted on a linear axis with error bars showing the arithmetic mean and 95% confidence interval. P values for dot plots were two-tailed and calculated using the Kruskal-Wallis test with Dunn’s multiple comparison correction. P values for reported sex versus neutralization were two-tailed and calculated by group using a two-way ANOVA with the Šidák multiple comparison correction. Scatter plots were prepared by first log transforming FRNT50 and EC50 data then performing simple linear fitting and plotting the 95% confidence bands. Correlations were calculated using Spearman’s correlation and two-tailed P values were calculated for the 95% confidence interval.

Acknowledgments

We acknowledge the study participants for their generous contributions; the OHSU COVID-19 serology study team and the OHSU occupational health department for recruitment and sample acquisition; and the OHSU clinical laboratory under the direction of Dr. Donna Hansel and Xuan Qin for SARS-Co-2 testing and reporting.
Funding: This study was funded by a grant from the M.J. Murdock Charitable Trust (MEC), an unrestricted grant from the Oregon Health & Science University (OHSU) Foundation (MEC), the National Institutes of Health training grant T32HL083808 (TAB), Oregon Health & Science University Innovative IDEA grant 1018784 (FGT), and National Institutes of Health grant R01AI145835 (WBM).
Author contributions: Conceptualization: TAB, HCL, ZLL, WBM, MEC, FGT. Cohort recruitment: ZLL, DS, BW, WBM, MEC. Sample acquisition and preparation: ZLL, DXL, DS, BW, JYL, WBM, MEC. Laboratory analysis: TAB, SKM, GG. Statistical analysis and visualization: TAB, JYL. Funding acquisition: TAB, WBM, MEC, FGT. Supervision: WBM, MEC, FGT. Writing – original draft: TAB. Writing – review and editing: all authors
Competing interests: The authors declare that they have no competing interests.
Data and materials availability: All data needed to support the conclusions of the paper are available in the paper or the supplementary materials.
This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. This license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using such material.

Supplementary Materials

This PDF file includes:

Figs. S1 to S4
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Other Supplementary Material for this manuscript includes the following:

MDAR Reproducibility Checklist
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La Comisión de Salud Pública matiza el tema de las terceras dosis tras infección: "El intervalo entre la infección y la administración de la dosis de recuerdo será de un mínimo de 4 semanas, pero se recomienda su administración a los 5 meses tras el diagnóstico de la infección"

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