Comparison of Levels of Nasal, Salivary, and Plasma Antibody to Severe Acute Respiratory Syndrome Coronavirus 2 During Natural Infection and After Vaccination

Abstract

Background

Most studies of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) measure antibody or cellular responses in blood; however, the virus infects mucosal surfaces in the nose and conjunctivae and infectious virus is rarely if ever present in the blood.

Methods

We used luciferase immunoprecipitation assays to measure SARS-CoV-2 antibody levels in the plasma, nose, and saliva of infected persons and vaccine recipients. These assays measure antibody that can precipitate the SAR-CoV-2 spike and nucleocapsid proteins.

Results

Levels of plasma anti-spike antibody declined less rapidly than levels of anti-nucleocapsid antibody in infected persons. SARS-CoV-2 anti-spike antibody levels in the nose declined more rapidly than antibody levels in the blood after vaccination of infected persons. Vaccination of previously infected persons boosted anti-spike antibody in plasma more than in the nose or saliva. Nasal and saliva anti-spike antibody levels were significantly correlated with plasma antibody in infected persons who had not been vaccinated and after vaccination of uninfected persons.

Conclusions

Persistently elevated SARS-CoV-2 antibody in plasma may not indicate persistence of antibody at mucosal sites such as the nose. The strong correlation of SARS-CoV-2 antibody in the nose and saliva with that in the blood suggests that mucosal antibodies are derived primarily from transudation from the blood rather than local production. While SARS-CoV-2 vaccine given peripherally boosted mucosal immune responses in infected persons, the increase in antibody titers was higher in plasma than at mucosal sites. Taken together, these observations indicate the need for development of mucosal vaccines to induce potent immune responses at sites where SARS-CoV-2 infection occurs.

Clinical Trials Registration

NCT01306084.

When SARS-CoV-2 vaccine was given intramuscularly to infected persons, the increase in antibody titers were higher in plasma than in the nose; antibody levels in the nose declined more rapidly than those in the blood. Mucosal vaccines are needed.

(See the Editorial Commentary by Edwards and Neuzil on pages 1400–2.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected >600 million persons worldwide and resulted in >6 million deaths. The virus enters the body through mucosal surfaces, particularly the nose and conjunctivae. Most studies have measured immune responses to the virus in the blood rather than at mucosal surfaces, such as the nose.

Antibody levels in blood have been shown to be correlated with protection from SARS-CoV-2 infection in nonhuman primates [1, 2] and in humans [3–8]. The presence of antibody to SARS-CoV-2 in blood, however, may not necessarily be correlated with the level of antibody at mucosal surfaces such as the nose and conjunctiva, at the site where infection occurs. Mucosal antibody can be derived from transudation of antibody from plasma or from plasma cells in the mucosa.

While hundreds of studies have measured antibodies to SARS-CoV-2 in the blood of infected persons or vaccine recipients, relatively few studies have evaluated antibodies in nasal secretions. Six studies have measured SARS-CoV-2 antibody levels in longitudinal samples from the nose and blood in persons who were infected [9, 10], vaccinated [11–13], or both [14, 15]. In the current study, we measured levels of antibody to SARS-CoV-2 in the nose, saliva, and plasma over time in a prospective study of persons infected with the virus before and after vaccination and in virus-naive study participants after vaccination.

METHODS

Study Participants and Specimens

Employees at the National Institutes of Health (NIH) in Bethesda, Maryland signed consents and were enrolled in a protocol approved by the NIH Institutional Review Board. Participants were enrolled and followed up from April 2020 to February 2022. Blood samples were obtained monthly from NIH Clinical Center healthcare personnel who had had ≥1 face-to-face patient contact. NIH employees, including healthcare workers, who had been infected with SARS-CoV-2 were eligible to participate if they had a polymerase chain reaction (PCR)–positive nasal swab sample and had recovered from infection. Severity of disease was based on the World Health Organization definition of severity [16]. Nasal fluid was obtained by inserting nasal synthetic absorptive matrix strips (Nasosorption FX-I; Hunt Developments) into each nostril and eluting the fluid. Additional details are available in the Supplementary Methods.

Antibody Assay

Antibodies to the spike and nucleocapsid proteins of SARS-CoV-2 were measured using the luciferase immunoprecipitation systems assay, as described elsewhere [17]. This assay measures antibody that immunoprecipitates fusion proteins containing the nucleocapsid protein or amino portion of spike protein, up to and including the receptor-binding domain. Additional details, including statistical methods, are available in the Supplementary Methods.

RESULTS

Demographics and Infectious Status of Study Participants

Between April 2020 and August 2021, the study enrolled 145 NIH Clinical Center healthcare personnel who had had ≥1 face-to-face patient contact; 91% were enrolled between April and July 2020 (Figure 1 and Supplementary Table 1). None had been vaccinated at the time of enrollment. The mean age of study participants was 46 years (range, 25–67 years), and 86% of participants were female. Half (50%) of the participants were nurses, 21% were therapists or food providers, 14% were physicians, physician assistants, or nurse practitioners, and 14% had other healthcare occupations. At the time of enrollment, 4 of the 145 healthcare personnel (3%) were SARS-CoV-2 seropositive for both anti-spike and anti-nucleocapsid antibody; 3 were asymptomatic, and 1 had had mild symptoms.

Figure 1.

Numbers of participants in the National Institutes of Health (NIH) healthcare worker and NIH-infected employee cohorts, including those from whom plasma samples were obtained, those who were vaccinated, and those from whom mucosal samples were obtained. Although all participants consented to provide blood samples, samples were not obtained from 4 participants in the first cohort (NIH healthcare workers) and 8 in the second cohort (Infected NIH employees). Abbreviation: PCR, polymerase chain reaction.

In addition, between May 2020 and August 2021, 83 NIH employees with a SARS-CoV-2–positive PCR result were enrolled after recovering from acute infection (Figure 1 and Supplementary Table 1); the median and mean intervals between symptom onset and enrollment were 58 and 93 days, respectively. The mean age of these participants was 43 years (range 22–72 years), and 66% were female. Most (83%) had mild symptoms without pneumonia or hypoxemia, 8.4% were asymptomatic, 4.8% had moderate symptoms with pneumonia but not markers of severe disease, and 3.6% had severe symptoms (respiratory rate >30, severe respiratory distress, or oxygen saturation <90% on room air). Of these PCR-positive employees, 78 (94%) were SARS-CoV-2 antibody positive; 3 of the 5 (60%) seronegative employees were asymptomatic; nasal fluid and saliva were not collected from these 5 employees.

Plasma Anti-Spike Antibody Responses to SARS-CoV-2 Compared With Anti-Nucleocapsid Antibody Responses in Infected Persons

Anti-nucleocapsid antibody levels declined in infected patients who had not been vaccinated, up to 400 days after onset of symptoms, while anti-spike antibody levels generally rose during the first 3 months after symptoms and then remained relatively constant through 400 days after symptom onset (Figure 2A and 2B ). The difference in the kinetics of the anti-spike and anti-nucleocapsid antibody titers was significant (P = .009). These findings may be related to the higher frequencies of memory B cells targeting spike protein observed in SARS-CoV-2–convalescent participants than memory B cells targeting nucleocapsid protein [18]. As expected, after vaccination of the infected persons, anti-spike antibody levels rose while anti-nucleocapsid antibody levels remained unchanged (Figure 2C and 2D ).

Figure 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plasma anti-nucleocapsid antibody levels decline, while anti-spike antibody levels rise and then remain stable in SARS-CoV-2–unvaccinated infected persons. A, B, SARS-CoV-2 antibody levels relative to time after the onset of symptoms in unvaccinated infected persons. C, D, Antibody levels relative to time before or after the first dose of vaccine. Anti-spike (A, C) and anti-nucleocapsid (B, D) antibody levels are displayed, as measured in light units (LUs). Each line represents an individual participant; horizontal dotted lines, cutoff levels for positive plasma anti-spike antibody and plasma anti-nucleocapsid antibody results (based on mean plus 3 and 4 standard deviations, respectively, for 32 uninfected blood donors); data represent 57 participants in A and B and 60 in C and D. The x axes show the time after the first dose of vaccine. The second dose of vaccine was given a median of 4 weeks after the first dose. The half-lives for anti-spike and anti-nucleocapsid antibody from the onset of symptoms to the first dose of vaccine were undefined (owing to the flatness of the curve) for anti-spike antibody (A, C) and >300 days for anti-nucleocapsid antibody (B, D). The half-lives after the second dose of vaccine were undefined (A) or 295 days (C) for anti-spike antibody and >300 days for anti-nucleocapsid antibody (B, D).

Declines in Nasal Versus Plasma Anti-Spike Antibody After Vaccination of Infected Persons

Because infection with SARS-CoV-2 begins at mucosal surfaces and the virus does not circulate in the blood [19], antibody present in nasal and oral fluids may be more relevant for protection against SARS-CoV-2 infection than antibody in the blood. In addition, immunoglobulin (Ig) A antibody is thought to be especially important for mucosal immunity. Therefore, we measured SARS-CoV-2 anti-spike antibody in the plasma, nose, and saliva as well as IgA-specific anti-spike antibody in infected NIH employees at multiple time points before and after vaccination. SARS-CoV-2 anti-spike and anti-nucleocapsid antibody levels were determined in fluid eluted from synthetic absorptive matrix strips placed in both the left and right nostrils. The levels of antibody were similar in both nostrils (Supplementary Figure 1); for subsequent analyses, results from the nostril with the highest level of antibody were used.

We next analyzed antibody decay after vaccination at time points beginning 1 week after the second vaccination. SARS-CoV-2 anti-spike antibody rapidly increased in the plasma of infected persons after vaccination and remained elevated for up to 300 days after vaccination (Figure 3A ). The level of nasal anti-spike antibody in infected persons after vaccination declined more quickly than that in plasma (P = .010). The levels of saliva anti-spike antibody and nasal anti-spike IgA antibody also declined after vaccination, although the differences between saliva anti-spike and plasma anti-spike antibody and between nasal anti-spike IgA antibody and plasma anti-spike antibody were not significant (P = .99 and P = .59, respectively). Thus, persistently elevated antibody levels in plasma may not reflect the same level of antibody persistence at mucosal sites such as the nose.

Figure 3.

Nasal and saliva severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) anti-spike antibody levels decline faster than plasma levels in infected persons after vaccination. Levels of anti-spike antibody in plasma, nasal fluid, and saliva and levels of immunoglobulin (Ig) A–specific anti-spike antibody, measured in light units (LUs), are shown for infected (A) and uninfected (B) persons after vaccination. Horizontal dotted lines represent cutoff level for positive plasma anti-spike antibody results. Sixty study participants were evaluated for plasma antibody and 37 for nasal and saliva antibody in A; 117 participants were evaluated for plasma antibody and 18 for nasal and saliva antibody in B. The x axes show the time after the first dose of vaccine. The second dose of vaccine was given a median of 4 weeks after the first dose. A, Half-lives for anti-spike antibody after the second dose of vaccine in infected participants were 295 days for plasma, 78 days for nasal fluid, 100 days for saliva, and 238 days for nasal IgA. B, Half-lives for anti-spike antibody after the second dose of vaccine in uninfected participants were undefined (owing to the flatness of the curve) for plasma, >300 days for nasal fluid, 220 days for saliva, and undefined for nasal IgA.

Evaluation of uninfected vaccinated persons showed that while plasma levels of SARS-CoV-2 anti-spike antibody remained high even 400 days after vaccination (Figure 3B ), antibody levels in the nose and saliva and IgA-specific antibody levels in the nose declined but were more variable. Fewer nasal and saliva samples from uninfected vaccinated persons were available for analysis, and the only significant differences noted were a more rapid decline in anti-spike antibody in saliva than in plasma (P = .02) and in nasal anti-spike IgA antibody versus saliva anti-spike antibody (P = .03).

Boosting of Plasma Versus Nasal Anti-Spike Antibody Beginning 1 Week After Vaccination of Infected Persons

We next compared the change in antibody levels in the plasma, nose, and saliva after vaccination of previously infected persons. Changes in antibody titers of 4-fold or higher are generally considered clinically meaningful. The level of SARS-CoV-2 anti-spike antibody in plasma increased 22.4-fold 1 week after the first dose of vaccine compared with prevaccination levels in previously infected persons (Figure 4). In contrast, the level of anti-spike antibody increased 14.1-fold in the nose and 12.3-fold in saliva after the first dose in these infected persons. The nasal anti-spike IgA level increased only 3.48-fold after vaccination. As a control, the level of anti-nucleocapsid antibody in plasma, the nose, or saliva changed little 1 week after the first vaccine dose: 0.45-fold, 0.85-fold, and 2.0-fold, respectively, in infected persons. Thus, a SARS-CoV-2 vaccine given intramuscularly boosted mucosal immune responses in infected persons, although the change in antibody titers in plasma was higher than at mucosal sites such as the nose and saliva.

Figure 4.

Lower ratio of anti-spike antibody in nose to that in blood in infected persons after the first vaccine dose compared with before the first dose. Figure displays fold changes in anti-spike and anti-nucleocapsid antibodies before the first dose and 1 week after the first dose of vaccine. Abbreviations: IgA, immunoglobulin A; NIH, National Institutes of Health; LUs, light units.

Significant Correlation Between Nasal and Saliva Anti-Spike Antibody Levels and Plasma Levels in Unvaccinated Infected Persons

Antibody in the nose and saliva may be due to transudation of the antibody from the blood or local production of antibody by tissue resident B cells or plasma cells. We determined whether antibody levels in the nose or saliva were correlated with plasma levels. In infected persons who had not been vaccinated, the level of anti-spike antibody in the blood was significantly correlated with levels in the nose (r = 0.356 and P < .001) and saliva (r = 0.285 and P < .001) (Figure 5A and 5B ). Similarly, the level of anti-spike antibody in the nose was significantly correlated with that in saliva (r = 0.562 and P < .001), although the correlation was less significant when levels of anti-spike IgA antibody in the nose were compared with anti-spike antibody in the plasma (r = 0.104 and P = .01) (Figure 5C and 5D ).

Figure 5.

A, B, Plasma severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) anti-spike antibody levels, measured in light units (LUs), are correlated with nasal (A) and saliva (B) anti-spike antibody levels in unvaccinated infected persons. C, D, Nasal SARS-CoV-2 anti-spike antibody is correlated with saliva anti-spike antibody (C), and plasma anti-spike antibody with nasal immunoglobulin (Ig) A anti-spike antibody (D) in unvaccinated infected persons. Data points represent results for individual participants (22 participants in A and C, 23 in B, and 21 in D), and vertical dotted lines represent cutoff levels for a positive plasma anti-spike antibody assay result.

In contrast, the levels of anti-nucleocapsid antibody in the nose (Supplementary Figure 2A) and in saliva (Supplementary Figure 2B) were not significantly correlated with the levels in the plasma in infected, unvaccinated persons (nasal vs plasma levels, r = 0.11 and P = .14; saliva vs plasma, r = 0.106 and P = .48). This lack of correlation may be related to relatively rapid decline in anti-nucleocapsid antibody over time (Figure 2B ). The strong correlation of SARS-CoV-2 anti-spike antibody levels in the nose and saliva with levels in the blood in infected, unvaccinated persons suggests that these antibodies were likely derived primarily from transudation from the blood rather than local production.

Significant Correlation Between Nasal and Saliva Anti-Spike Antibody Levels and Plasma Levels After Vaccination of Uninfected Persons

In uninfected persons who had been vaccinated, the level of SARS-CoV-2 anti-spike antibody in plasma showed significant correlation with levels in the nose (r = 0.639 and P < .001) and in saliva (r = 0.36 and P = .001) (Figure 6A and 6B ). The level of anti-spike antibody in the nose was correlated with that in saliva (r = 0.308 and P = .049) (Figure 6C ), and the level of anti-spike IgA antibody in the nose was significantly correlated with the level of anti-spike antibody in plasma (r = 0.284 and P < .001) (Figure 6D ).

Figure 6.

A, B, Plasma severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) anti-spike antibody levels, measured in light units (LUs), are correlated with nasal (A) and saliva (B) anti-spike antibody levels in uninfected vaccinated persons. C, D, Nasal SARS-CoV-2 anti-spike antibody is correlated with saliva anti-spike antibody (C), and nasal immunoglobulin (Ig) A anti-spike antibody with plasma anti-spike antibody (D) in uninfected vaccinated persons. Plasma was obtained ≥ 1 week after the second dose of vaccine. Data points represents results for individual participants (77 participants in A, 67 in B and C, and 74 in D).

In SARS-CoV-2–infected persons beginning 1 week after the second dose of vaccine, the level of SARS-CoV-2 anti-spike antibody in plasma also showed a significant correlation with that in the nose (r = 0.334 and P < .001) but a weaker correlation with that in saliva (r = 0.165 and P = .007) (Supplementary Figure 3A and 3B). The level of anti-spike antibody in the nose was significantly correlated with the level in saliva (r = 0.413 and P < .001) (Supplementary Figure 3C), while the level of anti-spike IgA antibody in the nose showed a weaker correlation with the level of anti-spike antibody in the plasma (Supplementary Figure 3D) (r = 0.119 and P = .001). The significant correlation between SARS-CoV-2 antibody levels in plasma and those in the nose and saliva after vaccination is consistent with the hypothesis that these mucosal antibodies result from transudation from the blood.

DISCUSSION

While most studies of antibodies in patients with SARS-CoV-2 have been performed using serum or plasma samples, mucosal responses are likely to be more important because the virus is transmitted by respiratory droplets or aerosol and infects the mucosa of the nose, oral cavity, and upper airway [20, 21]. Infectious virus is rarely if ever present in the blood [22, 23], except perhaps as a terminal event. We found marked differences between the levels of antibody in the nose and saliva and those in the blood, both during natural infection and after vaccination. SARS-CoV-2 antibody in the mucosa after natural infection may be associated with tissue resident memory B cells and local production of antibody by plasma cells from terminally differentiated plasma cells or short-lived plasmablasts [24] or transudation of antibody from the blood to mucosal surfaces. After intramuscular vaccination, neutralization of virus infection at the mucosal surface is thought to be due primarily to transudation of antibody from plasma [25]. While antibody in the blood has been shown to correlate with protection from SARS-CoV-2 infection in nonhuman primates and in humans, levels of antibody at mucosal surfaces are likely to be a more mechanistic correlate of protection.

In SARS-CoV-2–infected persons, we found that nasal antibody to the SARS-CoV-2 spike protein was induced with vaccination, but nasal antibody levels declined more rapidly than plasma antibody levels 1 week after the second vaccination. The more rapid fall in mucosal antibody levels may help explain the relatively high rates of reinfection seen in vaccinated persons despite the presence of antibody in the blood [26, 27]. In 1 report, the median interval between completion of vaccination and reinfection was only 86 days [28].

In a study that collected plasma and nasal swab samples from patients with coronavirus disease 2019 (COVID-19), during acute disease and about 3 and 8 months later, nasal levels of IgG and IgA antibody to the spike protein fell to almost undetectable levels at 3 months, while plasma levels of these antibodies remained elevated [14]. When several of these previously infected individuals were vaccinated and their antibody levels were measured twice, up to 6 weeks after the first dose of vaccine, the levels of IgG to the spike protein in nasal swab samples rose, and some were higher after vaccination than after infection. In contrast, vaccinated SARS-CoV-2–naive persons had much lower levels of antibody in nasal swab samples than vaccinated patients with COVID-19.

In a study looking at mucosal IgA in vaccinated healthcare workers who subsequently became infected, levels of nasal anti-spike mucosal IgA increased after infection [15]; however, unlike our study, the authors followed antibody levels for only 6 weeks after infection and did not follow the fall in these levels. While we also saw marked increases in mucosal antibody titers after vaccination of infected study participants, we focused our study on the rate of decline in mucosal and plasma antibodies in vaccinated participants, measuring antibody levels at multiple time points up to 42 weeks after vaccination. In a study of 17 persons, while nasal antibody to SARS-CoV-2 increased 1 week after onset of symptoms and declined between 1 and 9 months after infection, the antibody persisted at low levels for up to 9 months [10]. In a study of children 6 months after the diagnosis of COVID-19, fewer children had antibody to the virus in the nose than in the blood [9]. Thus, nasal antibodies decline more rapidly than plasma antibodies in infected persons.

In a study of 24 SARS-CoV-2 messenger RNA (mRNA) vaccine recipients who had previously not been infected, SARS-CoV-2 antibody levels declined more rapidly in the nose than in the blood [11]. SARS-CoV-2 antibody was detected in nasal swab samples from 90% of SARS-CoV-2 mRNA vaccine recipients 4 weeks after vaccination [13], while SARS-CoV-2 IgG was detected in both oral mucosal fluid and anterior nares samples from all recipients 90 days after the second dose of an mRNA vaccine [12].

We found that antibody to the SARS-CoV-2 spike protein was boosted more in the blood than in the nose beginning 1 week after vaccination of infected persons. This result suggests that vaccination at a nonmucosal site has a limited effect on protection from infection at mucosal sites and helps explain the need for frequent boosting at nonmucosal sites with the currently available vaccines.

We found that SARS-CoV-2 antibody levels in saliva and IgA in the noses of infected persons were boosted after vaccination. Previous studies have looked at SARS-CoV-2 antibody in saliva of naturally infected persons. Isho et al [29] found peak levels of IgG antibody to the virus in saliva 16–30 days after onset of symptoms, while IgA antibody declined rapidly and did not persist beyond day 60. IgG antibody in saliva was relatively stable up to 105 days after onset of symptoms.

We found that nasal and saliva antibody levels were significantly correlated with plasma levels in persons infected with SARS-CoV-2 who had not been vaccinated, and that nasal antibody and saliva anti-spike antibody levels were well correlated with plasma levels after vaccination of uninfected persons. These findings are consistent with the hypothesis that mucosal antibody is due to translocation from the blood. In a study of 28 patients with COVID-19, saliva IgG responses were significantly correlated with serum IgG responses [30], while in another study the correlation was modest [31]. In 2 other studies in which patients who recovered from COVID-19 were sampled at a single time point, serum IgG and IgA were correlated with nasal IgG and IgA [32, 33].

Vaccines given by the mucosal route, which resemble the route of natural infection with respiratory viruses, would be expected to produce more durable mucosal immunity than vaccines given intramuscularly [34]. Oral live attenuated rotavirus and poliovirus vaccines, as well as intranasal influenza, vaccines provide mucosal immunity. The oral poliovirus vaccine results in intestinal immunity with reduced shedding and less reinfection than the intramuscular poliovirus vaccine after exposure to wild-type poliovirus. In children, the intranasal live attenuated influenza vaccine results in approximately 50% better protection against disease compared with the inactivated intramuscular vaccine [35, 36] and induces longer-lasting antibody responses in both the blood and the nose [37]. Intranasal vaccination of animals with SARS-CoV-1, SARS-CoV-2, and Middle East respiratory syndrome vaccines showed that this route of vaccination was more effective against infection than intramuscular vaccination [38, 39]. Together with our findings of the more limited duration of SARS-CoV-2 antibody in the nose relative to the blood in infected persons and the higher levels of antibody in the blood than in the nose after intramuscular vaccination, this suggests that an intranasal vaccine for SARS-CoV-2 is more likely to provide a longer duration of protective antibody response at the site where infection occurs.

Supplementary Data

Supplementary materials are available at Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Notes

Author Contributions . J. I. C. had full access to all the study data and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: J. I. C., L. D., T. N. P., and P. D. B. Participant visits and data collection: L. D., K. G., K. M., K. L, . J. S. V., Y. L., and C. S. Sample preparation and antibody assays: K. W., T. K., M. A. A., P. D. B, and L. P. S.-W. Statistical analysis and drafting of the manuscript: J. Q. and J. W. Study supervision: J. I. C.

Disclaimer . The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government.

Financial support. This work was supported by the intramural research programs of the National Institute of Allergy and Infectious Diseases, the National Institute of Dental and Craniofacial Research, and the NIH Clinical Center and by the National Cancer Institute, National Institutes of Health (contract 75N91019D00024, task order no. 75N91019F00130).