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Salivary immune responses after COVID-19 vaccination
Longitudinal study to measure salivary immune responses after COVID-19 vaccination
https://orcid.org/0000-0003-1727-5441
Nguyen Kenny Data curation Formal analysis Methodology Writing – original draft 1
Relja Boris Investigation Methodology Writing – review & editing 1 2
https://orcid.org/0000-0003-1125-5247
Epperson Monica Investigation Methodology 3
Park So Hee Investigation Methodology 4
Thornburg Natalie J. Methodology Resources 3
Costantini Veronica P. Data curation Investigation Methodology Writing – review & editing 5
https://orcid.org/0000-0002-1530-3675
Vinjé Jan Conceptualization Project administration Supervision Writing – review & editing 5 *
1 National Foundation for the Centers for Disease Control and Prevention Inc., Atlanta, GA, United States of America
2 Cherokee Nation Assurance, Arlington, VA, United States of America
3 Laboratory Branch, Coronavirus and Other Respiratory Viruses Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, United States of America
4 Eagle Global Scientific, LLC, Atlanta, GA, United States of America
5 Division of Viral Diseases, Viral Gastroenteritis Branch, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, United States of America
Nasrallah Gheyath K. Editor
Qatar University, QATAR
Competing Interests: The authors have declared that no competing interests exist.

* E-mail: ahx8@cdc.gov
3 9 2024
2024
19 9 e03079368 12 2023
16 7 2024
https://creativecommons.org/publicdomain/zero/1.0/ This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

mRNA-based COVID-19 vaccines have played a critical role in reducing severe outcomes of COVID-19. Humoral immune responses against SARS-CoV-2 after vaccination have been extensively studied in blood; however, limited information is available on the presence and duration of SARS-CoV-2 specific antibodies in saliva and other mucosal fluids. Saliva offers a non-invasive sampling method that may also provide a better understanding of mucosal immunity at sites where the virus enters the body. Our objective was to evaluate the salivary immune response after vaccination with the COVID-19 Moderna mRNA-1273 vaccine. Two hundred three staff members of the U.S. Centers for Disease Control and Prevention were enrolled prior to receiving their first dose of the mRNA-1273 vaccine. Participants were asked to self-collect 6 saliva specimens at days 0 (prior to first dose), 14, 28 (prior to second dose), 42, and 56 using a SalivaBio saliva collection device. Saliva specimens were tested for anti-spike protein SARS-CoV-2 specific IgA and IgG enzyme immunoassays. Overall, SARS-CoV-2-specific salivary IgA titers peaked 2 weeks after each vaccine dose, followed by a sharp decrease during the following weeks. In contrast to IgA titers, IgG antibody titers increased substantially 2 weeks after the first vaccine dose, peaked 2 weeks after the second dose and persisted at an elevated level until at least 8 weeks after the first vaccine dose. Additionally, no significant differences in IgA/IgG titers were observed based on age, sex, or race/ethnicity. All participants mounted salivary IgA and IgG immune responses against SARS-CoV-2 after receiving the mRNA-1273 COVID-19 vaccine. Because of the limited follow-up time for this study, more data are needed to assess the antibody levels beyond 2 months after the first dose. Our results confirm the potential utility of saliva in assessing immune responses elicited by immunization and possibly by infection.

The author(s) received no specific funding for this work. Data AvailabilityAll relevant data are within the manuscript and its Supporting Information files.
OutbreaksCOVID-19
Data Availability

All relevant data are within the manuscript and its Supporting Information files.
==== Body
pmcIntroduction

Novel coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), became an ongoing global pandemic after being first reported December 2019 in Wuhan, China [1]. Since their emergency authorization for use, mRNA vaccines BNT162b2 and mRNA-1273 have been critical in limiting the spread of SARS-CoV-2 and preventing infected persons from becoming seriously ill from COVID-19 [2]. At the time of this study, initial regimens for these vaccines were intramuscularly in a two-dose schedule either 21 days (BNT162b2) or 28 days apart (mRNA-1273) [3, 4]. These mRNA vaccines consist of nucleoside-modified mRNA within a lipid nanoparticle to encode the spike (S) protein of SARS-CoV-2 [2]. Both vaccines induce neutralizing antibody titers against SARS-CoV-2, and initial clinical trials demonstrated 95% protection for BNT162b2 and 94.1% protection for mRNA-1273 against COVID-19 [5–8].

Antibody testing can be used to evaluate immune response from either vaccination or natural infection [9]. Traditionally, the gold standard method of antibody testing is through serology, as IgA, IgG, and IgM antibodies may be detectable in blood for several months or more after initial infection to the virus [10]. Systemic serum antibodies curtail virus propagation after infection of the host, whereas the presence of antigen-specific antibodies at the mucosal surfaces such as can be detected in the oral cavity is required to prevent initial infection of the host [11]. Although COVID-19 immune response has been extensively studied through serology, there are various logistical limitations from utilizing blood and serum as sample types [9]. For example, patients may be reluctant to participate due to pain or discomfort from the venipuncture collection method. Additionally, blood and serum samples must be collected by a healthcare professional, which could increase the chance of infection through direct contact [9, 12].

A non-invasive sample collection method such as using saliva can overcome some of the major practical challenges of serology testing and help increase patient participation [13]. Patients can self-collect their saliva samples at home and temporarily store samples in their home freezer at -20°C. Recent studies have confirmed that the antibody profile of IgG in saliva reflects that of serum [14], and these salivary assays can be utilized as a noninvasive alternative to blood to detect SARS-CoV-2-specific IgA and IgG with high sensitivity (IgA: 95.5%; IgG: 89.7%) and specificity (IgA: 99%; IgG: 97%) [15]. In addition, salivary antibody testing can also lead to novel insight on mucosal immune response against SARS-CoV-2 [16]. The use of salivary antibody tests as a supplement to serology can provide a robust data profile to help better understand the relationship between mucosal and systemic immunity to the virus to further support improved COVID-19 vaccines that initiates robust mucosal immune response shortly after exposure to SARS-CoV-2 [17].

The objectives of this study were to (i) evaluate SARS-CoV-2-specific IgA and IgG salivary immune response over time in participants who received 2 doses of the mRNA-1273 vaccine, (ii) examine duration and persistence of SARS-CoV-2-specific antibodies up to 6 months after the first dose and, (iii) compare the antibody kinetics in saliva and serum two weeks after the second vaccine dose.

Methods

Study design and saliva sample collection/processing

Between February 4, 2021 and September 4, 2021, participants working at the Centers for Disease Control and Prevention (CDC) were enrolled prior to receiving 2 doses (day 0 and a booster at day 28) of the mRNA-1273 SARS-CoV-2 vaccine. Participants were asked to self-collect saliva samples at the day of enrollment and at days 7, 14, 28, 42, 56 post immunization (dpi). Basic demographic information was collected from each participant. In addition to the self-collection of saliva, participants were invited to self-collect blood at day 42 (14 days after receiving their second dose of mRNA-1273 vaccine).

Saliva samples were collected by passive drool method [18] using the SalivaBio Collection Aid (SCA; Salimetrics, Carlsbad, CA). Participants were provided saliva collection kits (Salimetrics, Carlsbad, CA), which consisted of a disposable SCA that securely fits within an included microtube to streamline and simplify sample collection. Email reminders were sent to participants on their collection dates to ensure accurate and timely sample collection. Saliva was collected at least 30 minutes after consumption of food or liquids by passively drooling into the collection aid which flowed directly into the attached microtube. Saliva was stored in each participant’s home freezer (-20°C) until day 56. Once received in the laboratory, saliva samples were γ-irradiated (2 x 106 rads) to inactivate possible SARS-CoV-2 and were clarified by centrifugation (5 min. at 10,000 x g) and stored at -80°C until testing for SARS-CoV-2 IgA and IgG antibodies.

Enzyme immunoassay (EIA) to detect salivary IgA and IgG antibodies against SARS-CoV-2

IgA and IgG salivary antibodies against SARS-CoV-2 spike protein were detected as described previously [15]. Briefly, Immunolon 2 HB flat 96 well plates (Fisher Scientific) were coated with 100 μL / well of SARS-CoV-2 spike protein (Wuhan-Hu-1 strain, GenBank MN908947.3) (0.5 μg/mL) or, to normalize against total IgA or IgG, with goat anti-human IgA (α-chain) or IgG (g-chain) (0.5 μg/ml) in Dulbecco’s phosphate buffered saline (DPBS) and incubated overnight at 4°C in a humidified chamber. Positive control (SARS-CoV-2 convalescent serum), negative control (pre-pandemic saliva samples collected from healthy adults (2009–2010) [19], and blank controls were included in each run. Plates were washed and blocked using blocking buffer (DPBS 1X Tween-20/5% skimmed milk) for 2 hrs. at 37°C. After washing, 4-fold serial dilutions (1:10, 1:40, 1:160) of each saliva sample were added to both SARS-CoV-2 spike protein and DPBS-coated wells. Bound antibodies were detected using HRP-conjugated goat anti-human IgA 1:4,000 (Sera Care) or goat anti-human IgG 1:16,000 (Sera Care) diluted in blocking buffer for 1 hr at 37°C. After washing, 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Sera Care) was added for 5 min. and the reaction was stopped by adding 100 μL of Stop solution (Sera Care). Plates were read at 450 nm and 630 nm using an Epoch2 instrument (BioTek) and upon background correction, the optical density (OD) values were calculated (OD450nm−OD630nm).

Multiplex electrochemiluminescent immunoassay for SARS-CoV-2 blood testing

Capillary blood samples were self-collected by participants using Mitra Neoteryx microsampling kits according to the manufacturers’ instructions and were stored at -20°C. Serum was recovered by eluting one microsampler tip (20 μl) in 400 μl of 1x PBS (Gibco) + 1.0% BSA (Sigma) + 0.5% Tween-20 (Sigma) in a 1 ml deep-well 96 well plate (ThermoFisher). Plate was sealed with an adhesive foil seal and incubated overnight at 4°C on a shaker at 300 rpm. The resulting eluate was stored at -80°C until use. Antibody testing was performed as a 3-plex assay using the V-PLEX SARS-CoV-2 Panel 2 (IgG) multiplex serology assay (Meso Scale Diagnostics, MD) according to the manufacturer’s instructions [20]. This kit quantifies antibodies to three SARS-CoV-2 antigens including spike protein (S), nucleocapsid (N), and S1 receptor binding domain (RBD) of SARS-CoV-2 spike protein. Antibody concentrations are interpolated from a standard curve, calibrated by the manufacturer to the WHO 1st International Reference Standard (NIBSC 20/136) and reported in binding antibody units per mL (BAU/mL). Serum eluates were evaluated at two dilutions using the manufacturer’s (kit) diluent (final in-plate dilutions 1/5,000 and 1/50,000). Briefly, plates were blocked for 30 minutes at room temperature on an orbital shaker (700 rpm) and washed three times with kit wash buffer using an automated plate washer (Agilent, CA). Diluted samples were added to the plate along with assay standards and controls and incubated for 2 h at room temperature on an orbital shaker (700 rpm). After washing three times with kit wash buffer, bound antibodies were detected by incubating with SULFO-TAG labeled anti-human IgG antibody for 1 h at room temperature on an orbital shaker (700 rpm). After washing three times, MSD read buffer was added and plates were read on a Meso Sector S 600 instrument. Raw data were processed using MSD Discovery Workbench v4.0 and final summaries and data compilation created using the SAS software Enterprise Guide v7.11 (SAS Institute, Cary, NC).

Data analysis

To quantify both total and SARS-CoV-2 specific antibodies, a standard curve for IgA or IgG was prepared by serial dilution of purified human IgA or IgG [15]. Adjusted OD values were plotted against concentration and fitted to a sigmoidal 4 parameter logistic model. To account for participant differences (e.g., severity of illness, immunocompetency, collection time, antibody secretion levels), SARS-CoV-2 specific IgA or IgG were normalized to 100 μg of total IgA or IgG, respectively. When virus-specific antibodies could not be detected, but total IgA or IgG antibodies were detected, SARS-CoV-2-specific IgA (or IgG) were arbitrarily assigned as half the lower limit of detection, based on the standard curve of purified human IgA (IgG) [15]. SARS-CoV-2-specific salivary IgA or IgG titers were extrapolated from the linear portion of the standard curve. For saliva samples, median and interquartile ranges (IQR) were used for descriptive statistics of SARS-CoV-2 antibody levels. Kruskal-Wallis test followed by Dunn’s comparison was performed to assess significant differences between titers obtained from different days after vaccination. Statistical analyses were performed in GraphPad Prism 9.

For serum samples, binding antibody units (BAU) per mL were captured and plotted for 3 different SARS-CoV-2 antigens: nucleocapsid (N) protein, receptor binding domain (RBD), and spike (S) protein. The following manufacturer’s positivity cutoff values were used for N, RBD, and S, respectively: 11.8 BAU/mL, 14.6 BAU/mL, and 17.7 BAU/mL. Boxplots were constructed in GraphPad Prism 9.

Ethics statement and disclaimer

This project was approved by CDC (project ID: 0900f3eb81c8ea60) and was conducted consistent with applicable federal law and CDC policy (See, e.g., 45 C.F.R. part 46.102(l)(2), 21 C.F.R. part 56; 42 U.S.C. §241(d); 5 U.S.C. §552a; 44 U.S.C. §3501 et seq.). Written informed consent was obtained from each participant. The findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention (CDC).

Results

In this study, 203 CDC staff members were enrolled prior to receiving two doses of the mRNA-1273 vaccine (Fig 1). Of these, 3 participants were excluded due to voluntary withdrawal. Overall, 1023 saliva samples were collected from 200 participants between day 0 and day 56. The median number of samples collected per day was 161.5 (range 155–200). All samples were tested for SARS-CoV-2 specific IgA and IgG antibodies. Additionally, 23 participants provided an extra sample 6 months after the first dose for SARS-CoV-2 specific IgG antibody detection. Basic demographic information from the participants is listed in Table 1.

10.1371/journal.pone.0307936.g001 Fig 1 Study design and sample collection.

A total of 1046 saliva samples were collected from 200 participants who received the two-dose mRNA-1273 vaccine. Participants self-collected saliva samples at 6 different time points after their first COVID-19 vaccine dose: day 0 (first dose–baseline), 7, 14, 28 (second dose), 42, 56 and 6 months. Samples were stored in participants’ home freezers at -20°C until drop-off at the laboratory for testing.

10.1371/journal.pone.0307936.t001 Table 1 Demographic summary of enrolled participants (n = 200).

Age (y)	Sex (%)	Race (%)	Ethnicity (%)	
Range	23–68	Female	127 (64%)	White	127 (64%)	Not Hispanic or Latino	179 (90%)	
Mean	43.294	Male	73 (36%)	Black or African American	35 (17%)	Hispanic or Latino	15 (8%)	
				Asian	32 (16%)	Not reported	6 (2%)	
				Other	6 (3%)			
				American Indian or Alaskan Native	0 (0%)			
				Native Hawaiian or Other Pacific Islander	0 (0%)			

IgA titers exhibited a sharp rise two weeks post the first vaccine dose, followed by a rapid decline after four weeks. Salivary IgA titers significantly increased between days 0 and 14 (p<0.0001), day 7 and 14 (p<0.0001), and day 28 and day 42 (p = 0.0059) (Fig 2A). Conversely, IgG titers increased two weeks after the first dose (p<0.0001) while no further increase in titers on day 28 was observed (Fig 2B). Salivary IgG titers further increased through day 56 (Fig 2). Significant differences among IgG titers were observed two weeks after the first dose (day 14; p<0.0001) and two weeks after the second dose (day 42); p<0.0001) (Fig 2).

10.1371/journal.pone.0307936.g002 Fig 2 SARS-CoV-2-specific IgA and IgG salivary antibody profiles upon receiving mRNA-1273 vaccine.

To evaluate SARS-CoV-2-specific immune responses from IgA and IgG, all 1023 saliva samples were tested using our in-house enzyme immunoassay. SARS-CoV-2 antibodies were normalized to 100 μg of total salivary IgA or IgG, respectively to account for differences among participants, collection time and secretion levels. Log transformation was performed on the data set. Log transformed results for normalized IgA (A) and IgG (B) were plotted against each saliva collection timepoint: day 0, 7, 14, 28, 42, and 56. Negative samples have been arbitrarily assigned a value of -1. Boxes represent 25th percentile, median, and 75th percentile, and the whiskers show the 2.5–97.5 percentile. Kruskal-Wallis test followed by Dunn’s multiple comparisons test was performed. Ns (p>0.05); * (p<0.05); ** (p<0.01); ***(p<0.001); **** (p<0.0001). Arrows indicate first and second vaccine dose.

SARS-CoV-2 IgA and IgG antibody titers between age groups, gender and race were analyzed and showed no significant difference (S1–S3 Figs).

To better understand the persistence of salivary IgG immune response, saliva samples were collected from 23 participants 6 months after the first vaccination dose. Compared to day 56, the IgG titers remained elevated, without significant difference, albeit slightly lower (p = 0.0954) (Fig 3).

10.1371/journal.pone.0307936.g003 Fig 3 Longitudinal salivary antibody profile of SARS-CoV-2-specific IgG in 23 study participants at an extended collection timepoint.

To evaluate the persistence of salivary IgG antibody trends, saliva samples from 23 subjects were collected at 6 months post first dose. Results were normalized by dividing the SARS-specific Ig by the total Ig. A log transformation was performed on the data set, and negative samples were arbitrarily assigned a value of -1. Boxes represent 25th percentile, median, and 75th percentile, and the whiskers show the 2.5–97.5 percentile. Kruskal-Wallis test followed by Dunn’s multiple comparisons test was performed. ns (p>0.05); * (p<0.05); ** (p<0.01); ***(p<0.001); **** (p<0.0001). Arrows indicate first and second vaccine dose.

In addition to saliva, capillary blood samples from 127 consenting participants were collected at day 42 (14 days after receiving the second dose of mRNA-1273 vaccine). All participants showed a strong IgG response to both the RBD and S (spike) protein, whereas 12 (9.4%) participants had antibodies against the nucleocapsid proteins. The median antibody titer against the RBD protein was 14786.35 BAU/ml, 9943.57 BAU/ml against the spike protein, and 3083.92 BAU/ml against the nucleocapsid protein (Fig 4).

10.1371/journal.pone.0307936.g004 Fig 4 Multiplex serology assay results for spike, nucleocapsid, and RBD proteins in blood samples (n = 127) collected 42 days after the first vaccine dose.

A box plot was generated to visualize the distribution of antibody levels for each protein. The y-axis represents the binding antibody levels arbitrary units, with the horizontal line within each box representing the median value. IgG levels were measured against 3 different SARS-CoV-2 antigens: RBD, S, and N. A separate sub-plot was constructed for the N antigen to better visualize results.

Discussion

There is increasing recognition that the mucosal immune system and local secretory neutralizing antibodies in oral fluids and nasopharyngeal fluids may be a barrier to reinfection as well as an impediment to SARS-CoV-2 shedding and transmission [11, 21]. In addition, saliva is preferred by health care workers as an appealing alternative to phlebotomy as it can be self-collected, is non-invasive and cheaper to acquire [22]. In this study, we determined salivary immune responses from CDC staff members receiving 2 doses of the mRNA-1273 vaccine. All participants developed detectable salivary antibodies after receiving the 2-dose regimen, a response similarly observed in other studies [23, 24]. SARS-CoV-2-specific IgA titers peaked 2 weeks after each vaccine dose, albeit administration of the second dose of vaccine boosted the IgG but not the IgA response which rapidly declined, a trend that mirrors findings reported for antibodies against the spike protein in serum [25, 26]. This suggests that the mRNA-1273 vaccine can elicit a rapid IgA response in saliva of vaccinated individuals. Salivary IgG antibody titers significantly increased 2 weeks post first dose, reached a peak 2 weeks after the second dose and thereafter stayed elevated until at least day 56, when the last saliva sample was collected. For the 23 participants for which saliva was collected 6 months after the first vaccine dose, salivary IgG titers remained elevated as has been reported by others [23, 27]. These findings suggest that, compared to venipuncture blood collection, detection of salivary antibodies, in particular IgG, may be a useful alternative to evaluate immune responses to COVID-19 vaccination [23, 28]. Other studies analyzing SARS-CoV-2-specific IgA and IgG in serum and saliva of infected patients found similar patterns, with IgA levels increasing after infection followed by a rapid decline whereas IgG levels maintained a sustained, delayed response post-infection [10, 29]. However, recent reports suggest that saliva is less sensitive than serum for the detection of IgG antibodies following vaccination and therefore cannot be used interchangeably post vaccination or infection [30, 31]. Although we focused on mucosal immune responses after immunization rather than natural infection, our results showed similar persistence patterns of SARS-CoV-2-specific salivary IgG up to 6 months [32].

Our study has several limitations. First, since we only tested serum samples from a subset of 127 participants for nucleocapsid antibodies, previous exposure to infection with SARS-CoV-2 of the remaining 73 participants during the time of the study cannot be ruled out. We detected nucleocapsid antibodies in 9.4% at day 42 post vaccination, an indication of an infection either prior to the enrollment of the participant or during the study. Most published studies have been shown that nucleocapsid antibodies can be detected at least for 11 months after SARS-CoV-2 infection [33, 34]. However, recent data have shown that that in non-exposed participants salivary immune responses after SARS-CoV-2 infection may develop much later [35], which could have been addressed by testing blood samples beyond day 42 after vaccination. Therefore, our overall conclusions on the kinetics of the immune responses after vaccination should be taken with caution. Second, our study population was relatively small reducing the potential generalizability of the findings and limit their statistical power. In addition, our results do not represent the race or ethnicity distribution in the USA. We also did not consider other factors that may impact the salivary immune response to COVID-19 vaccination, such as the presence of comorbidities or use of certain medications that may affect immune functions or previous infection history [36]. Finally, we did not evaluate whether salivary antibodies did neutralize SARS-CoV-2, which could provide more conclusive information on immune protection against the virus [37, 38].

Our study demonstrates that the Moderna mRNA-1273 COVID-19 vaccine mounts a salivary SARS-CoV-2 IgA and IgG immune response. Further studies are needed to determine the potential correlations between salivary antibody levels and vaccine effectiveness [39]. Our results confirm the potential utility of saliva in assessing immune responses elicited by immunization and/or natural infections.

Supporting information

S1 Table Individual salivary IgA and IgG titers.

(XLSX)

S1 Fig SARS-CoV-2 salivary IgA and IgG titers after mRNA-1273 vaccination—comparison between <60 years old and 60+ years old age groups.

(TIF)

S2 Fig SARS-CoV-2 salivary IgA and IgG titers after mRNA-1273 vaccination—comparison by gender.

(TIF)

S3 Fig SARS-CoV-2 salivary IgA and IgG titers after mRNA-1273 vaccination–comparison by race.

(TIF)

We are grateful to the participants for their willingness to participate during the challenging time of the pandemic. We thank Owen Herzegh and David Petway at the Division of Scientific Resources for gamma irradiation of the saliva samples prior to antibody testing. We also thank David Holmes and Holly Ann Williams at the CDC’s Occupational Health Clinic and Michael Beach and Sherrie Bruce at the National Center for Emerging and Zoonotic infectious Diseases for their logistical support during the vaccine study enrollment process.

10.1371/journal.pone.0307936.r001
Decision Letter 0
Nasrallah Gheyath K. Academic Editor
© 2024 Gheyath K. Nasrallah
2024
Gheyath K. Nasrallah
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is an interesting study whose objective was to evaluate the salivary IgA and IgG immune response after vaccination with the COVID-19 Moderna mRNA-1273 vaccine. Their results confirm the potential utility of saliva in assessing immune responses after immunization and highlights that testing could lead to novel insight on mucosal immune response against SARS-CoV-2.

MINOR REVISION:

The infection status of the individuals before or after vaccination is not mentioned along the writing. Figure 4 is showing reactivity to nucleocapsid for some individuals and in Figure 3, saliva samples showed positivity at day 0 and 7, indicating that some patients had viral exposure within or before the study period.

Because SARS-CoV-2 infections occurring before, between or after doses could influence the level and persistence of anti-SARS-CoV-2 IgA or IgG antibodies in saliva and blood specimens, it would be advisable to incorporate this information into the study.

Additionally, it would be important to conduct analyses comparing individuals who experienced infections with those who did not, to recognize if there are any significant differences. Or at least justify why it is not shown.

Reviewer #2: Dear authors, please address the following points:

- line 111 authors states they used pre-pandemic saliva samples as negative control, please clarify the source of these sample, where they came from a bank of saliva sample collected from generic patients before pandemic? How were they stored?

-line 125 authors wrote:"participants self-collected whole blood", in my opinion it is worthy specify that the collected sample are Capillary blood sample;

- line 132 remove one square bracket;

- line 174 the total number of 1023 seems wrong. (200-42)*6=948. If you excluded 42 patients from the original 200, it means you analyzed 158 patients. So the total number of collected samples should be 948.

- line 178. In table 1 you should report information on the 158 patients really enrolled in the study excluding information of patients excluded.

- line 186 The dot at the end of the line is a typo. A dot instead should be inserted at the end of line 187.

- line 191 Authors stated they collected blood samples from 127 partecipants, was this blood collected from a venipuncture procedure? Please clarify.

- line 194 and line 254. Authors analyzed data reporting median and percentile, meaning data are not distributed normally. But they performed ANOVA test, that is usually used for data distributed normally. Can authors please clarify and justify the statistical test performed?

Reviewer #3: The manuscript reports induction of salivary antibody titers upon vaccination against SARS-CoV-2. Authors report significant increase in anti-Spike IgA and IgG two weeks after vaccination. It is a bit surprising, given that the development of antibody response takes around 2-3 weeks. This suggests that antibody response detected in these patients is driven by memory B cells, but not by naive B cells. The data on the status of participants are required: were these donors previously infected? Figure 4 reports some of the data that can be used rto determine the status, but these data are measured at day 42 after vaccination. It is of particulary importance for the data interpetation, since we have recently shown that in non-exposed participants salivary immune responses develop mcuh later (Bondareva et al., Cell Host and Microbe, 2023). Also, authors do not provide the levels of toal IgA/IgG in saliva. These parameters may vary quite extensively in saliva during collection and the normalisation to total IgA/IgG levels may further show whether this effect due to increase of specific antibodies or total antibody levels (bondrave et al., J of autoimmunty, 2022).

Minor comments:

1. Authors describe in the introduction the salivary Ab measurement as easier way to estimate blood Ig levels, but do not really mention that Ab in the oral cavity are of physiological significance, since they do actually protect against the initial virus infection. I would add this in introduction with the relevant quotes.

2. Authors do not cite a lot of relevant literature that was focused on salivary Ab responses, including series of works from J. Gommermann, and others. Thus, discussion should be extended and should include all the relevant papers published on this topic. So far it is quite poor and ignores many seminal works addressing the kinteic and importance of these responses.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Eleonora Nicolai

Reviewer #3: Yes: andrey kruglov

**********

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10.1371/journal.pone.0307936.r002
Author response to Decision Letter 0
Submission Version1
14 Apr 2024

RESPONSE TO EDITOR/REVIEWER COMMENTS

We would like to thank the reviewers for their critical review of our manuscript and their helpful comments to more clearly describe the data leading to a significantly improved revised manuscript. Many thanks for your time!

Reviewer #1: This is an interesting study whose objective was to evaluate the salivary IgA and IgG immune response after vaccination with the COVID-19 Moderna mRNA-1273 vaccine. Their results confirm the potential utility of saliva in assessing immune responses after immunization and highlights that testing could lead to novel insight on mucosal immune response against SARS-CoV-2.

MINOR REVISION:

The infection status of the individuals before or after vaccination is not mentioned along the writing. Figure 4 is showing reactivity to nucleocapsid for some individuals and in Figure 3, saliva samples showed positivity at day 0 and 7, indicating that some patients had viral exposure within or before the study period. Because SARS-CoV-2 infections occurring before, between or after doses could influence the level and persistence of anti-SARS-CoV-2 IgA or IgG antibodies in saliva and blood specimens, it would be advisable to incorporate this information into the study. Additionally, it would be important to conduct analyses comparing individuals who experienced infections with those who did not, to recognize if there are any significant differences. Or at least justify why it is not shown.

RESPONSE: Healthy participants without respiratory symptoms were enrolled immediately after they received their first mRNA vaccine dose and although we asked if they had experienced symptoms that could be associated with COVID-19, most of them declined to provide that info and no nurses were available to collected a day 0 blood sample and hence the only lab data we have on their infection status is a saliva sample at day 0.

Reviewer #2: Dear authors, please address the following points:

Comment #1: - line 111 authors state they used pre-pandemic saliva samples as negative control, please clarify the source of these sample, where they came from a bank of saliva sample collected from generic patients before pandemic? How were they stored?

RESPONSE: Negative saliva samples were collected in 2009-2010 from healthy adults and stored at -80°C. We have modified to include this information and a reference to the original study (line 114 in the revised manuscript)

Comment #2-line 125 authors wrote: "participants self-collected whole blood", in my opinion it is worthy specify that the collected sample are Capillary blood sample;

RESPONSE: Thanks. We updated that capillary blood samples were collected.

Comment #3- line 132 remove one square bracket;

RESPONSE: bracket removed

Comment #4- line 174 the total number of 1023 seems wrong. (200-42)*6=948. If you excluded 42 patients from the original 200, it means you analyzed 158 patients. So the total number of collected samples should be 948.

RESPONSE: We have reviewed the data and realized that the statement in line 174 is incorrect. A total of 203 participants were enrolled, and 3 dropped out the study. Not all participants provided 6 samples (day 0-56). Overall, 1023 saliva samples were collected. Six months after the first dose, 23 participants provided an additional saliva sample. Hence, a total of 1046 saliva samples were collected, tested and analyzed. We have corrected the manuscript as follows:

Line 31: Two hundred was replaced as “…Two hundred three…”

Line 174 (line 118 in revised version): We replaced “200” by “203”

Line 175 (line 182 in revised version): We replaced “42” by ”3” and deleted the statement “or inability to complete the entire sample collection series through day 56.”

Line 176 (line 183-184): We have updated the sentence to reflect the number of saliva samples collected per day and the total number of saliva samples. “Overall, 1023 saliva samples were collected from 200 participants between day 0 and day 56. The median number of samples collected per day was 161.5 (range 155-200).”

Line 179 (185-186): rephrased to ”Additionally, 23 participants provided an extra sample 6 months after the first dose for SARS-CoV-2 specific G antibodies detection”

Figure 1: The 6 months’ time-point was included as well as the number of saliva samples collected for each time point. The legend was also updated.

Comment #5- line 178. In table 1 you should report information on the 158 patients really enrolled in the study excluding information of patients excluded.

RESPONSE: We are sorry for the misunderstanding. As explained for comment #4 above, data from 200 participants were analyzed.

Comment #6- line 186 The dot at the end of the line is a typo. A dot instead should be inserted at the end of line 187.

RESPONSE: Removed and added a dot were indicated.

Comment #7- line 191 Authors stated they collected blood samples from 127 participants, was this blood collected from a venipuncture procedure? Please clarify.

RESPONSE: “Blood samples was replaced by “Capillary blood sample”

Comment #8- line 194 and line 254. Authors analyzed data reporting median and percentile, meaning data are not distributed normally. But they performed ANOVA test, that is usually used for data distributed normally. Can authors please clarify and justify the statistical test performed?

RESPONSE: Upon reviewing your concern about the use of ANOVA for data reported as median and percentiles, we realized that the final analysis was conducted using unpaired t-tests. The error in the manuscript was due to the fact that we initially attempted ANOVA analyses but later switched to non-parametric tests due to our data characteristics, sample size/distribution, and comparative analyses of the different sub-groups. Relevant sections have been corrected in the revised manuscript.

Reviewer #3: The manuscript reports induction of salivary antibody titers upon vaccination against SARS-CoV-2. Authors report significant increase in anti-Spike IgA and IgG two weeks after vaccination. It is a bit surprising, given that the development of antibody response takes around 2-3 weeks. This suggests that antibody response detected in these patients is driven by memory B cells, but not by naive B cells.

The data on the status of participants are required: were these donors previously infected? Figure 4 reports some of the data that can be used to determine the status, but these data are measured at day 42 after vaccination. It is of particular importance for the data interpretation, since we have recently shown that in non-exposed participants salivary immune responses develop much later (Bondareva et al., Cell Host and Microbe, 2023).

RESPONSE: see response to reviewer #1 above. Healthy participants without respiratory symptoms were enrolled immediately after they received their first mRNA vaccine dose and although we asked if they had experienced symptoms that could be associated with COVID-19, most of them declined to provide that info and no nurses were available to collected a day 0 blood sample and hence the only lab data we have on their infection status is a saliva sample from each participant collected at day 0.

Also, authors do not provide the levels of total IgA/IgG in saliva. These parameters may vary quite extensively in saliva during collection and the normalization to total IgA/IgG levels may further show whether this effect due to increase of specific antibodies or total antibody levels (Bondareva et al., J of autoimmunty, 2022).

RESPONSE: Thank you for pointing that out as we did normalize SARS-CoV-2 titers to total IgA and IgG and have now included additional language to the manuscript. Lines 157-160 in the revised manuscript. To account for participant differences (e.g., severity of illness, immunocompetency, collection time, antibody secretion levels), SARS-CoV-2 specific IgA or IgG was normalized to 100 µg of total IgA or IgG, respectively. When virus-specific antibodies could not be detected, but total IgA or IgG antibodies were detected, SARS-CoV-2-specific IgA (or IgG) were arbitrarily assigned as half the lower limit of detection, based on the standard curve of purified human IgA (IgG) (Costantini et al., 2022).

Minor comments:

Authors describe in the introduction the salivary Ab measurement as easier way to estimate blood Ig levels, but do not really mention that Ab in the oral cavity are of physiological significance, since they do actually protect against the initial virus infection. I would add this in introduction with the relevant quotes.

RESPONSE: Thank you. Added to the introduction.

Authors do not cite a lot of relevant literature that was focused on salivary Ab responses, including series of works from J. Gommermann, and others. Thus, discussion should be extended and should include all the relevant papers published on this topic. So far it is quite poor and ignores many seminal works addressing the kinetic and importance of these responses.

RESPONSE: Thank you very much. We have added additional relevant papers on salivary antibody responses in the Discussion.

Attachment Submitted filename: REBUTTAL manuscript PONE-D-23-39898.docx

10.1371/journal.pone.0307936.r003
Decision Letter 1
Nasrallah Gheyath K. Academic Editor
© 2024 Gheyath K. Nasrallah
2024
Gheyath K. Nasrallah
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version1
30 Apr 2024

PONE-D-23-39898R1Salivary immune responses after COVID-19 vaccinationPLOS ONE

Dear Dr. Vinjé,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

While we appreciate the effort you put into revising your manuscript based on the reviewers' feedback, I regret to inform you that we are unable to accept the manuscript for publication at this time. One of the major comments raised by one of the reviewers has not been adequately addressed in the revised version. I cannot proceed with the publication until all major concerns raised by the reviewers have been satisfactorily resolved.

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We look forward to receiving your revised manuscript.

Kind regards,

Gheyath K. Nasrallah

Academic Editor

PLOS ONE

Additional Editor Comments:

While we appreciate the effort you put into revising your manuscript based on the reviewers' feedback, I regret to inform you that we are unable to accept the manuscript for publication at this time. One of the major comments raised by the reviewers has not been adequately addressed in the revised version. I cannot proceed with the publication until all major concerns raised by the reviewers have been satisfactorily resolved.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: No

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Dear Editor, in my opinion the authors have addressed all the questions raised and now the paper is suitable for publication.

Best regards

Reviewer #3: As it became evident during revision, the previous exposure to SARS-CoV-2 by participants cannot be ruled out. Thus, the conclusions about the kinetics of immune response upon vaccination cannot be made. In my opinion, this is a crucial point of the paper. Authors could have critically evaluated their data based on the already published reports, where clinical status was well documented. However, they also did not provide proper discussion in the revised manuscript and do not cite relevant literature.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Eleonora Nicolai

Reviewer #3: No

**********

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

10.1371/journal.pone.0307936.r004
Author response to Decision Letter 1
Submission Version2
21 Jun 2024

RESPONSE TO EDITOR/REVIEWER COMMENTS – PONE-D-23-39898R2

We would like to thank the reviewers for their critical review of our manuscript and their helpful comments to more clearly describe the data leading to a significantly improved revised manuscript. Many thanks for your time!

Reviewer #2:

Dear Editor, in my opinion the authors have addressed all the questions raised and now the paper is suitable for publication.

Answer: Thank you

Reviewer #3:

As it became evident during revision, the previous exposure to SARS-CoV-2 by participants cannot be ruled out. Thus, the conclusions about the kinetics of immune response upon vaccination cannot be made. In my opinion, this is a crucial point of the paper. Authors could have critically evaluated their data based on the already published reports, where clinical status was well documented. However, they also did not provide proper discussion in the revised manuscript and do not cite relevant literature.

Answer: We expanded on the limitation paragraph of our manuscript as we cannot rule out previous exposure to SARS-CoV-2 infection. We expanded on the data of the 9.4% of participants who tested positive for nucleocapsid antibodies 42 days after vaccination and thus the 90.6% who didn’t have serum antibodies, and emphasized that our conclusion on the kinetics of the immune responses after vaccination should be taken with caution and rephrased the paragraph as follows:

Our study has several limitations. First, since we only tested serum samples from a subset of 127 participants for nucleocapsid antibodies, previous exposure to infection with SARS-CoV-2 of the remaining 73 participants during the time of the study cannot be ruled out. We detected nucleocapsid antibodies in 9.4% at day 42 post vaccination, an indication of an infection either prior to the enrollment of the participant or during the study. Most published studies have been shown that nucleocapsid antibodies can be detected at least for 11 months after SARS-CoV-2 infection [33, 34]. However, recent data have shown that that in non-exposed participants salivary immune responses after SARS-CoV-2 infection may develop much later [35], which could have been addressed by testing blood samples beyond day 42 after vaccination. Therefore, our overall conclusions on the kinetics of the immune responses after vaccination should be taken with caution. Second, our study population was relatively small reducing the potential generalizability of the findings and limit their statistical power. In addition, our results do not represent the race or ethnicity distribution in the USA. We also did not consider other factors that may impact the salivary immune response to COVID-19 vaccination, such as the presence of comorbidities or use of certain medications that may affect immune functions or previous infection history [36]. Finally, we did not evaluate whether salivary antibodies did neutralize SARS-CoV-2, which could provide more conclusive information on immune protection against the virus [37, 38].

Attachment Submitted filename: Response to Reviewers PONE-D-23-39898R2.docx

10.1371/journal.pone.0307936.r005
Decision Letter 2
Nasrallah Gheyath K. Academic Editor
© 2024 Gheyath K. Nasrallah
2024
Gheyath K. Nasrallah
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version2
16 Jul 2024

Salivary immune responses after COVID-19 vaccination

PONE-D-23-39898R2

Dear Dr. Vinie,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Gheyath K. Nasrallah

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: All the comments have been addressed. The manuscript can be accepted for the publication in Plos One.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: Yes: Andrey Kruglov

**********

10.1371/journal.pone.0307936.r006
Acceptance letter
Nasrallah Gheyath K. Academic Editor
© 2024 Gheyath K. Nasrallah
2024
Gheyath K. Nasrallah
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
22 Jul 2024

PONE-D-23-39898R2

PLOS ONE

Dear Dr. Vinjé,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

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on behalf of

Dr. Gheyath K. Nasrallah

Academic Editor

PLOS ONE
==== Refs
References

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2 Teo SP . Review of COVID-19 mRNA Vaccines: BNT162b2 and mRNA-1273. J Pharm Pract. 2021:8971900211009650. Epub 2021/04/13. doi: 10.1177/08971900211009650 .33840294
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