Saliva as a useful tool for evaluating upper mucosal antibody response to influenza.

Mucosal immunity plays a crucial role in controlling upper respiratory infections, including influenza. We established a quantitative ELISA to measure the amount of influenza virus-specific salivery IgA (sIgA) and salivary IgG (sIgG) antibodies using a standard antibody broadly reactive to the influenza A virus. We then analyzed saliva and serum samples from seven individuals infected with the A(H1N1)pdm09 influenza virus during the 2019-2020 flu seasons. We detected an early (6-10 days post-infection) increase of sIgA in five of the seven samples and a later (3-5 weeks) increase of sIgG in six of the seven saliva samples. Although the conventional parenteral influenza vaccine did not induce IgA production in saliva, vaccinated individuals with a history of influenza infection had higher basal levels of sIgA than those without a history. Interestingly, we observed sIgA and sIgG in an asymptomatic individual who had close contact with two influenza cases. Both early mucosal sIgA secretion and late systemically induced sIgG in the mucosal surface may protect against virus infection. Despite the small sample size, our results indicate that the saliva test system can be useful for analyzing upper mucosal immunity in influenza.

Introduction

Influenza A virus (IAV) causes an acute upper respiratory disease in humans, resulting in seasonal influenza and occasional severe lung diseases of zoonotic origin [1]. Because of the ever-mutating IAV genome, the IAV vaccine is updated every year based on global surveillance and the clinical data on the circulating virus strains during the winter season in the opposite hemisphere [2]. Still, influenza causes almost 650,000 deaths worldwide every year [3].

The situation with influenza has dramatically changed because of the emergence of a novel coronavirus, SARS-CoV-2, in late 2019. The COVID-19 pandemic, caused by SARS-CoV-2 infection, has overwhelmed influenza-related fatalities since early 2020 [4]. However, seasonal IAV still exists among humans. Therefore, after the COVID-19 pandemic is brought under control by SARS-CoV-2 vaccination, the number of influenza cases in association with close human-to-human contact may rise again in the absence of any protective measures [4].

Although current influenza vaccines do not induce a sterilized immunity, they are likely to be effective in reducing the severity of influenza [2]. The most of the current influenza vaccines are administered parenterally. They elicit systemic IgG antibody response against IAV, but little mucosal IgA antibody, and therefore, are insufficient to prevent infection at mucosal surfaces [5]. Because it has been shown that mucosal IgA and IgG antibodies largely contribute to the protection against influenza virus infection [6], a nasal route of immunization is considered more effective for offering protection from upper respiratory infections [5]. Importantly, a nasal immunization can induce secretory IgA that forms dimers or even multimers and exhibits more potent and broader neutralizing activity than serum IgA monomers [7].

An intranasal vaccine containing inactivated whole virions has been demonstrated to be immunogenic and safe since the early 2000s. However, a licensed intranasal vaccine has not been widely utilized [8]. In the US, the nasal spray flu vaccine consisting of a live attenuated IAV (LAIV) was approved in 2003; however, its application is limited to healthy non-pregnant individuals aged 2–49 [9]. In the UK, a quadrivalent LAIV was introduced for children in the 2013–2014 flu season; it is now recommended for children aged 2–17 years [10]. Notably, the effectiveness of the live IAV vaccine so far does not surpass the more widely used and safer inactivated IAV vaccine [11, 12]. In Japan, an intranasal vaccine formula of inactivated IAV was developed and may be available for all ages [13]. It will be interesting to know how efficacious this intranasal vaccine can be.

To measure mucosal anti-IAV responses after intranasal IAV vaccine administration, nasal or nasopharyngeal wash samples have been collected [13, 14]. However, collecting nasal or nasopharyngeal washes is not easy practice and unsuitable for mass sampling. Thus, a simpler method to evaluate the mucosal immune responses is desired for developing an intranasal IAV vaccine [14]. The saliva sampling is much safer, easier, and less painful than nasal/nasopharyngeal sampling; in fact, saliva tests have long been recognized as sensitive assays of viral antibodies [15]. Furthermore, the detection rate of respiratory viruses in saliva and nasopharyngeal aspirates are shown to be comparable [16]. Therefore, saliva collection could be an alternative to nasal wash collection for evaluating the mucosal status after a virus infection.

As SARS-CoV-2 is frequently transmitted during meals and face-to-face conversations and then replicated in the organs in the oral cavity [17], a saliva test is suitable for detecting SARS-CoV-2 RNA via RT-PCR [18, 19] and comparable with nasopharyngeal swabs [20]. Thus, saliva sampling is valuable for detecting both viral RNA and host mucosal antibody responses in COVID-19 [21]. Moreover, detecting serum and saliva antibodies will enhance the serological study of SARS-CoV-2 infection [22, 23] and even other upper respiratory infections. In this context, Russel et al. have claimed that the evaluation of mucosal and serum IgA response is important for the understanding the asymptomatic and mild states of COVID-19 [24].

In this study, we evaluated the usefulness of saliva samples for detecting mucosal immune responses against IAV infections. With regards to the antibody class, Waldman et al. detected IgG and IgA, but not IgM, in bronchoalveolar lavage fluids and nasal washings after mucosal immunization of inactivated IAV vaccine [25]. In a common-cold coronavirus infection, it was shown that IgG and IgA, but not IgM, can persist for extended periods in the serum and nasal fluids [26]. Therefore, we here focused on only IgA and IgG antibodies. We established a sandwich ELISA system to measure the amount of influenza virus-specific IgA and IgG in saliva and serum. We tested the samples collected from individuals diagnosed with IAV infection from December 2019 to February 2020 and healthy individuals before and after IAV vaccination. We showed here that saliva samples were potentially valuable for evaluating immune responses to influenza virus infection, especially salivary IgA (sIgA) response at an early time point and salivary IgG (sIgG) response at a later time point. With non-invasive and safe sampling, a large-scale serological screening system using saliva would be possible to study asymptomatic cases with various upper respiratory virus infections or evaluate intranasal vaccines.

Materials & methods

1. Sample collection

Every year, we have been recruited student and staff volunteers from the Division of Medical Technology, Tokyo University of Technology, and obtained their serum and saliva samples with written informed consent before the influenza season (pre-stocks). During the 2019–2020 flu season from December 2019 to early February 2020, right before various measures for preventing SARS-CoV-2 transmission were widely promoted in Japan [27], there were 15 cases of influenza. They were diagnosed with IAV infection via a rapid flu test kit in local clinics using nasal swab samples. We have no influenza cases since then. Among the 15 cases, 7 complete sets of saliva samples, including the samples before infection (pre-serum), at approximately 6–10 days (early), after the documented infection (1 day before the onset of high fever was set at day 0), and at 3–5 weeks after infection, were available (Table 1, down to no.2013). Unfortunately, the pre-serum samples were unavailable in 5 of 7. The serum and saliva samples of two additional individuals were also analyzed, who had close contact with individual no. 2013 (B in Table 1) just before the influenza onset; one infected no. 2010 and the other asymptomatic no.2014 (A and C in Table 1, respectively).

Table 1

Saliva sample information of A(H1N1)pdm influenza virus-infected individuals in the 2019–2020 flu season.

Donor ID	Gender	Age (years)	Pre (month in 2019)	Influenza infection (month, year)	Early p.i.	Late p.i. (weeks)	Influenza history	2019/20 vaccine
2003	F	20	11	12. 2019	day 8	5	No	No
2005	F	20	11	12. 2019	day 6	>3	No	Yes
2006	M	20	11	12. 2019	day 6	>3	No	No
2007	M	20	11	12. 2019	day 9	4	No	No
2008	F	20	11	12. 2019	day 8	4	Yes	Yes
2016	M	22	11	2. 2020	day 8	4	No	No
2013 (B)	F	21	11	1 2020	day 8	5	Yes	Yes
2010 (A)	F	21	na	1. 2020	day 8	5	No	No
2014 (C)	F	21	na	asymptomatic	-	*	No	Yes

na, not available; p.i., post-infection (1 day before the onset of high fever was set at day 0).

*, the samples were collected at the same time with A and B.

Also, we used the saliva samples of 11 individuals who received an influenza vaccine during the 2018–2019 flu season completely different from influenza-infected individuals; the samples were stocked before (pre) and approximately 1 month after (post) vaccination (Table 2).

Table 2

Saliva Sample information of vaccinated individuals in the 2018–2019 flu season.

Donor ID	Gender	Age	Pre (month. year)	Post-vaccination (weeks)	Influenza history	Vaccination history
19V1	M	20	10. 2018	4	Yes	Yes
19V2	F	21	10. 2018	4	Yes	Yes
19V3	F	21	11. 2018	4	Yes	No
19V4	F	21	11. 2018	4	No	No
19V5	F	21	11. 2018	4	No	Yes
19V6	F	22	11. 2018	6	No	No
19V7	F	22	11. 2018	6	No	No
19V8	F	22	11. 2018	8	No	Yes
19V9	M	67	11. 2018	5	No	Yes
19V10	M	66	11. 2018	>3	No	Yes
19V11	M	21	11. 2018	4	Yes*	Yes

*Infected with influenza B virus in February 2018.

Saliva samples were collected using SalivaBio (Salimetris, CA, USA) oral swabs and centrifuged at 1,710 × g for 15 min, and the supernatants were transferred to new tubes. All the samples were kept in a −80°C freezer until use. The study followed the Helsinki declaration and was approved by the ethical committee of the Tokyo University of Technology (No. E18HS-023).

2. Reagents and viruses

An inactivated influenza virion of A(H1N1)pdm09 vaccine strain [A/Singapore/GP1908/2015 (H1N1)] was kindly provided by Dr. Takeshi Tanimoto (BIKEN Co. Ltd., Kagawa, Japan). The live H1N1 (A/Singapore/GP1908/2015) and H3N2 (A/Hong Kong/4801/2014) viruses were obtained from the Influenza Virus Research Center, National Institute of Infectious Diseases (NIID). These viruses were expanded in MDCK (ATCC) cells, and their respective titers were determined using the hemagglutination activity (HA) assay with type O human red blood cells (RBCs). The H1HA, HA protein of H1N1 (A/Singapore/GP1908/2015) virus was produced by a baculovirus expression system (Thermo Fisher Scientific, MA, USA).

3. Generation of standard influenza-specific antibodies

FI6 is a well-known, broadly IAV-reactive human monoclonal antibody [28]. The expression plasmids of the VH and LH gene of the FI6-IgG antibody were constructed using a recombinant technology with a human antibody expression cassette [29]. The constant region of the IgA heavy chain gene cloned from the peripheral blood mononuclear cells of a healthy donor was used to generate an FI6-IgA-expression plasmid by replacing the genes encoding the constant region of IgG with that of IgA using Gibson assembly (New England Biolab., MA, USA). The recombinant FI6-IgG and FI6-IgA antibodies were produced using the Expi293TM Expression System Kit (Thermo Fisher Scientific). FI6-IgG and FI6-IgA were purified with Protein G Sepharose 4B (Thermo Fisher Scientific) and Peptide M/agarose (InvivoGen Inc., San Diego, CA, USA), respectively.

4. Hemagglutination inhibition (HI) test

The HI test was performed using live H1N1 (A/Singapore/GP1908/2015) and H3N2 (A/Hong Kong/4801/2014) viruses according to the standard protocol provided by WHO (https://www.who.int/influenza/gisrs_laboratory/cnic_serological_diagnosis_hai_a_h7n9_20131220.pdf) with slight modifications. In summary, each serum was first pretreated with RDE to remove non-specific hemagglutinin inhibitors and pre-adsorbed with type O human RBCs. Then, the sera were serially diluted 1:2 starting from a 1:20 diluted solution with 25 μL/well in a 96-well conical-bottom plate (Watson Bio Lab, Tokyo, Japan). Next, the diluted sera were incubated with four HA influenza virus titers at 25 μL/well for 1 h at room temperature (RT: 25°C) and then with 0.75% of human RBCs at 50 μL/well for 1 h at 4°C. The minimum HI titer was 20.

5. ELISA

The amount of total IgA in saliva was measured. First, a Nunc 96-well microtiter plate (Thermo Fisher Scientific) was coated with a mouse monoclonal anti-human-IgA antibody (22C) [30] at 1 μg/mL and kept at 4°C overnight. The next day, the plate was washed, blocked with PBS/1% BSA at RT for 1.5 h before the samples were added. The samples were first serially diluted 5-fold with PBS/1% BSA/0.1% Tween-20 starting from a 1:100 dilution solution. As a standard, purified myeloma IgA1 protein (Sigma-Aldrich Inc., Tokyo, Japan) was used in 1:2 serial dilutions starting from 20 ng/mL. Then, the plate was placed at 4°C overnight, washed again, and incubated with a biotinylated anti-human IgA antibody (Southern Bio-Tech, Birmingham, UK). After a 1-h incubation at RT, the plate was washed and reacted with HRP–streptavidin (1:2000 dilution with PBST, BioLegend) for 30 min. Afterward, the TMB substrate (Sigma-Aldrich) was added after washing. Last, the reaction was stopped and measured at OD450 using a microplate reader iMARKTM (BioRad, Hercules, CA, USA).

The amount of influenza-specific IgA and IgG in each sample was measured using the aforementioned ELISA protocol, except the plate was coated with inactivated A(H1N1)pdm09 virions (1:2000 dilution) or H1HA protein (2 μg/mL) in PBS. Purified recombinant FI6-IgA or FI6-IgG antibody was used as a standard to measure virion-specific IgA or IgG. In some experiments, a purified human IgG against H1HA was used to measure H1HA-specific IgG. Saliva samples were diluted serially 1:3 starting from a 1:20 dilution solution and serum samples were diluted serially 1:5 starting from a 1:100 dilution solution. The secondary antibody used was a biotinylated anti-human IgA or IgG antibody (Southern Bio-Tech).

6. Statistical analysis

The amount of A(H1N1)-specific IgA and IgG antibodies was calculated on the basis of a standard curve of FI6-IgA and FI6-IgG, respectively, using Microplate Manager 6 (BioRad). Statistical analysis was performed using GraphPad Prism version 9. The differences between groups were analyzed using the nonparametric Mann–Whitney test or Wilcoxon matched-pairs test. A P-value <0.05 was considered statistically significant.

Results

1. HI titer and anti-H1HA IgG antibodies in the serum

From December 2019 to early February 2020, the A(H1N1)pdm09 influenza virus caused a major epidemic in Japan; however, A(H3N2) influenza infections were rare [23]. Therefore, we first conducted the HI assay to analyze the serum of seven individuals diagnosed with IAV infection (Table 1) to determine the viral strain. Although the pre-serum was unavailable in five of the seven individuals, we detected >80 HI titers against A(H1N1)pdm09 in all the late (3–5 weeks after infection) samples except no. 2008 (Fig 1A., * no sample available). Notably, donor nos. 2003, 2006, 2007, and 2016 were never vaccinated. In contrast, donor nos. 2005, 2008, and 2013 were vaccinated every year, including the 2019–2020 flu season. Concerning the HI titer against the A(H3N2) influenza virus, it was found <40 in all the serum samples.

Fig 1

The titer of Hemagglutination inhibition (HI) and serum IgG in influenza cases in the 2019–2020 flu season.

Serum samples of seven donors diagnosed with influenza A virus infection were analyzed. (a) Standard HI assay. The y-axis shows the HI titers. (b) ELISA. The amount of IgG was calculated on the basis of the standard curve of a purified H1HA-specific human IgG antibody. Grey columns and symbols, before infection; blank columns, 8–10 days after infection (early); black columns and symbols, >1 month after infection (late).

We also measured the amount of H1HA [A/Narita/1/2009(H1N1)]-specific serum antibodies via ELISA. The results were roughly consistent with those of HI titers; also, a slight increase in the level of specific IgG was detected in donor no. 2008 (Fig 1B). Because the “pre” serum was available only in no. 2016 and no. 2013, it is not clear whether the relatively high basal level of HA-specific serum IgG in no. 2016 is within the variability of ELISA measurement or due to some other reasons. Collectively, the data suggest that all seven individuals were infected with the A(H1N1)pdm09 influenza virus.

2. The influenza-specific IgA increases earlier than the influenza-specific IgG in saliva

Because the level of secretory IgA in the saliva is high and known to vary over a day [28], we simultaneously measured the total amount of sIgA and calculated the portion of influenza-specific IgA. The amount of total sIgA varied from 11.8 to 65.5 μg/mL, and the ratio of the early and late samples to the pre sample was 0.261–4.274. The portion of influenza-specific sIgA increased at the early sample in five (nos. 2007, 2016, 2005, 2008 and 2013) of the seven individuals and decreased significantly later, based on the pre-infection titers (fold increase in Fig 2A, right panel). In contrast, the fold increase of specific sIgG tended to be higher later (Fig 2B) than early. Notably, in the individuals (nos. 2003 and 2006) who showed little increase in sIgA even in the late time point, the level of sIgG increased especially in the late time point (Fig 2A and 2B, right panels). To make the time course difference clearly visible, the fold sIgA and sIgG increases were summarized according to the early and late time points in Fig 2C. Although the later decline of sIgA was not significant (p = 0.078), the level of sIgG was significantly increased later (p = 0.031).

Fig 2

The level of saliva antibodies in A(H1N1)pdm influenza virus-infected individuals.

Total sIgA, A(H1N1)pdm virion-specific sIgA, and sIgG were determined using ELISA. (a) The calculated amount of specific sIgA (ng) per 1 μg of total sIgA is depicted on the y-axis. Right panel: the fold increases from pre-antibody titers. (b) The y-axis indicates the amount of specific sIgG (ng). Right panel: the fold increases from pre-antibody titers. Grey column, before infection (pre); blank column, 8–10 days after infection (early); black column, 3–5 weeks after infection (late). (c) The fold sIgA and sIgG increases in the right panels were summerized according to the early and late time points. *P < 0.05.

3. No increase of the influenza-specific salivary IgA in vaccinated individuals

Parenteral vaccination does not significantly induce mucosal IgA responses [5]. We verified this observation by analyzing the A(H1N1)pdm09-specific sIgA in 11 vaccinated individuals (Table 2) before (pre) and after (post) inoculation with the 2018–2019 flu vaccine. A very low level of A(H1N1)pdm09-specific sIgA was detected (Fig 3A, left panel); the sIgA levels remained largely unchanged after vaccination except in V11. The mean titers in all the pre and post samples were similar, at 0.485 and 0.504 ng/μg total sIgA per milliliter, respectively. In some donors, the titer was even decreased after vaccination. Thus, basically, the sIgA was not increased by vaccination. Of note, the basal level of specific sIgA tended to be higher in three individuals (Group A: V01–V03) who had a previous history of influenza >3 years ago compared with that in the seven individuals (Group B: V04–V10) who had no influenza history. Because donor V11, who was infected with an influenza B virus 9 months before vaccination, displayed a considerable increase in the level of sIgA after vaccination, we excluded the person from the comparison between Groups A and B. Nevertheless, the sIgA levels were significantly higher in Group A than those in Group B in both the pre- and post-vaccination samples (p < 0.033 and p < 0.022, respectively) (Fig 3, right panel).

Fig 3

The level of saliva antibodies in vaccinated individuals.

The saliva samples were collected before (pre) and after (post) the 11 individuals received the influenza vaccine. Total sIgA and A(H1N1)pdm-specific sIgA were determined using ELISA, and influenza virus-specific sIgA (ng) per total sIgA (μg) was calculated as described in Fig 2. Right panel: the difference between Group A (n = 3) with a previous history of influenza and Group B (n = 7) without the history of influenza was depicted. Donor V11, who had a recent influenza B virus infection, was excluded from the comparison. Grey column, before infection (pre); black column, 3–5 weeks after infection (late). *P < 0.05.

4. Symptomatic and asymptomatic infection in contact cases

Finally, we present an example of infection caused by the close contact among three female students. One student (A) developed influenza in the first day of January, and the other (B) developed influenza 1 day later. Both were diagnosed with influenza A. The third individual (C) never developed any symptoms. The level of serum H1HA-specific IgG was increased in A and B (Fig 4A), whereas it was already high in C before the incidence and only increased slightly later.

Fig 4

Serum and saliva antibodies in close contact cases.

(a) The level of serum anti-H1HA IgG before (pre) and 1 month after infection incidence (late) in three donors. (b) The level of sIgA (left) and sIgG (right) of these individuals (ng/mL). Grey columns: before infection (pre); blank columns, 8–10 days after infection (early); black columns, >1 month after infection (late). *No sample was available.

The levels of sIgA and sIgG were measured in these three individuals (Fig 4B). The saliva sample of C was collected with that of A and B only at the later time point. Although A’s pre-sample was unavailable, the level of A(H1N1)pdm09-specific sIgA was remarkably higher in A than in B at the early time point (Fig 4B, left).

Interestingly, C had a substantial level of sIgA after the contact incidence. A similarly high level of sIgG was detectable in all individuals at the late time point (Fig 3B, right). A had never been vaccinated, whereas B and C were vaccinated every year. Of note, C received a flu vaccine just 1 week before the incident, and her pre-serum sample was obtained 1 month before the vaccination. Therefore, the slight increase in the level of serum IgG in C at the late time point could be due to the vaccination. Conversely, a substantial level of sIgA may reflect a transient exposure to an influenza virus.

Discussion

We showed the usefulness of saliva as a biomarker of the mucosal immune status during an upper respiratory infection by analyzing the sIgA and sIgG responses to influenza virus infection. We collected samples from individuals who were clinically diagnosed with influenza type A and confirmed that they were infected with the A(H1N1)pdm09 influenza virus. Since those patients are treated with an anti-influenza drug within 48 h after the onset of symptoms, saliva samples may not fully reflect the natural course of influenza virus infection. Nevertheless, we could detect the early increase (at 6–10 days post-infection) of sIgA and later increase (at 3–5 weeks) of sIgG. The mucosal immune system consists mainly of nasopharynx-associated lymphoid tissues (NALT) and gut-associated lymphoid tissues (GALT) and is partly compartmentalized depending on the actual route of induction [24]. It is noted that parotid secretory IgA in saliva could be linked to immune induction in tonsils/adenoid (human NALT) and cervical lymph nodes than that in GALT [31]. In contrast, most IgG in saliva has been considered to be derived from the blood circulation by passive leakage [31]. In fact, the plasma and salivary IgG profiles are shown to be highly similar [32]. Therefore, a later increase of sIgG likely reflects a systemic IgG response. Thus, despite the small sample size, this is the first important study indicating that saliva sampling is quite valuable for the analysis of mucosal anti-influenza response in parallel with a systemic response.

The level of secretory IgA is one of the key parameters in evaluating mucosal vaccination. The representative mucosal antibodies after intranasal vaccination can be obtained by nasal washing with 100 mL of PBS [13, 14], a non-invasive but unpleasant and even painful procedure. Thus, nasal wash sampling is unsuitable as a routine procedure for ordinary people. In contrast, saliva collection is simple, easy, and painless. In addition, analyzing salivary IgA represents an easy method of measuring immunogenicity after administering intranasal vaccination with LAIV to children [33]. Furthermore, oral fluid sampling has been recently validated for assessing responses to LAIV vaccine [34]. Our data indicate that saliva can serve as a simple indicator of mucosal immune responses to upper respiratory virus infections. Moreover, saliva tests may help evaluate and develop mucosal vaccines [8, 14, 35].

Parenteral vaccination is considered to induce little IgA in the mucosa, including saliva [8]. Consistent with this notion, the current parenteral flu vaccine did not induce A(H1N1)pdm09 virus-specific IgA in the saliva in this study. One exception was donor V11 (Fig 3), who was infected with influenza B virus in February 2019, 9 months before flu vaccination. As antibodies against the A(H1N1)pdm09 influenza virus persist for at least 15 months [36], the residual mucosal immune response against some common antigens of influenza viruses may be reactivated through vaccination. Interestingly, the basal level of A(H1N1)pdm09 virus-specific IgA was relatively higher in the three donors with a previous history of influenza than that in the seven donors without a history of influenza. Thus, the result suggests that while the subcutaneous vaccination failed to induce mucosal antibody responses, the primed individuals by a previous IAV infection can maintain the mucosal antibody to some extent. However, the levels of influenza-specific IgA are too low for us to conclude. Further study with larger sample size is necessary.

The protective role of mucosal IgA against upper respiratory infections has long been contested by challenge studies using animal models and humans [37]. In fact, the A(H1N1) challenge study by Gould et al. of individuals with only low HI titers showed that H1N1 specific IgG levels did not correlate with protection; meanwhile, specific IgA levels shortened the duration of virus shedding, indicating that IgA contributed to protection against influenza [38]. By analyzing mucosal antibody responses in saliva of three individuals who were simultaneously exposed to the influenza virus one (case C in Fig 4) of the three persons received vaccination a week ago and remained asymptomatic. Her serum IgG level appeared to be already high compared with that of others when they were exposed to an influenza virus. Interestingly, both sIgA and sIgG responses were detected in this individual at the late time point after the exposure. A high level of sIgA might indicate that the mucosal antibodies are induced by brief exposure to an influenza virus and the serum IgG induced by vaccination reflects the level of sIgG as demonstrated by Hettegger et al. that plasma and salivary anti-hepatitis B IgG profiles are highly similar for each individual infected with hepatitis B virus [32]. However, it remains unclear here which mucosal IgA and IgG antibodies play a major protective role. The analysis of more contact infection cases may shed light on the protective effect of mucosal antibody responses at an early phase of upper respiratory infections.

The induction and longevity of IgA-type antibody-secreting and memory B cells in the upper respiratory virus infection remains poorly understood. Interestingly, in the study of respiratory syncytial virus (RSV) challenge, Habiti et al. detected influenza-specific IgA memory B cells in the peripheral blood circulation [39]. Because humans are not naïve to IAV infection, it will be challenging to study the generation/evolution of upper respiratory memory IgA B-cell responses in influenza. The recently emerged SARS-CoV-2 virus infection may provide a good opportunity to clarify this issue. In this context, a wave of IgA plasmacells in the blood is considered to occur prior to the production of secretory IgA antibodies, with a peak at around 6–10 days after mucosal infection/immunization [24]. Recently, Sterlin et al. reported that early SARS-CoV-2-specific humoral responses were dominated by IgA antibodies and IgA plasmablasts with mucosal homing potential was detected early [40]. It is an interesting question to address how and where such mucosal homing IgA plasmablasts and memory cells are induced and maintained in humans.

Conclusions and perspectives

Serum antibody responses are mostly measured in serological studies in epidemiology or evaluations of protection by vaccines. However, in respiratory infections, which occur primarily in the upper respiratory mucosa, both mucosal and serum antibody responses should be measured in parallel. Many questions remain unanswered in the study of influenza. For example, how frequently do asymptomatic influenza infections occur? How are influenza viruses maintained in humans during summertime? Most importantly, what is the true correlate of protection in vaccination? Large-scale serological studies will be critical for answering these questions. In that sense, our results strongly support the notion that measuring salivary IgA and IgG in addition to serum IgG is a promising way of mass screening the intranasal vaccination status or identifying asymptomatic infections of influenza and other respiratory viruses including SARS-CoV-2 [24]. The usefulness of saliva has already been noted in SARS-CoV-2 infection and other virus infections. How saliva testing contributes to understanding the human respiratory immune responses needs to be studied further.

(XLSX)

Click here for additional data file.

19 Oct 2021

29 Nov 2021

Responses to reviewers

Reviewer #1: The theme has scientific relevance and has a well-structured methodology, unfortunately it has a small sample size, however, this does not minimize the merit of the study.

The introduction presents the topic clearly and objectively. The methodology is adequate. The presentation of the results is clear, however it presents poor quality figures. Regarding the discussion, this could be more clear and fluid in reading, the paragraphs are without continuity of reasoning. The first paragraph could be more aimed at presenting the novelty in the study's findings, with fewer descriptions and without going back to introductory or methodological issues. The literature review seems adequate enough to support the discussion.

Overall, a structural review of the discussion could enrich the presentation of this study and consequently improve its quality.

- Thank you for your kind and constructive comments. We have uploaded the figures with better resolution and added a new Fig.2c.

- We completely agree with you that the first paragraph has too much redundancy. We have extensively reorganized the discussion part. We hope that you find now the appropriate presentation of our study in the discussion of the revised manuscript.

Reviewer #2: According to the authors, the objective of this study, in a general way, was to evaluate the potential use of saliva to measure the amount of influenza virus-specific IgA and IgG antibodies by using a quantitative ELISA "in house". Interesting data were presented and they can support the putative use of saliva samples to monitor the influenza virus infection or vaccination by using the quantitative ELISA test purposed by the authors. However, it is necessary to clarify that the main limitation of the study was the low number of samples evaluated. In addition, there are another couple of factors that should be concerned, too.

- Thank you very much for your helpful comments. We admit the limitation of this study on the low number of samples. However, these samples are valuable especially because we were able to use “pre” saliva samples before A(H1N1)pdm influenza infection. They are essential to evaluate the fold antibody increase at early and late time points. As reviewer #1 commented, we believe that a small sample size will not minimize the merit of the study, and our results are worth publishing. We have claimed this point in the last part of first paragraph in the discussion.

In the Abstract section:

1) Please describes the salivary IgA and IgG as secretory, which allows us to differ from serum IgA and IgG.

- Thank you for your suggestion. To discriminate serum and saliva antibodies, we use sIgA and sIgG for salivary antibodies in the abstract and a whole text.

2) Please state in which sample the results concerning early IgA and latter IgG were found. It is not clear if these results were observed only in saliva or only in serum or in both fluids.

- Thank you for your comment. Our major finding using saliva samples is illustrated in Fig. 2, where the fold increase of sIgA (Fig. 2a) and sIgG (Fig. 2b) were depicted at the right panels. As you see, early increase (blank column) was observed in 5 of 7 individuals, whereas in all individuals except no. 2015, the fold increases of sIgG were higher at later (filled column) than early time point. To make the time course difference clearly visible, the fold sIgA and sIgG increases were summarized according to the early and late time points in Fig. 2c (new).

3) Since IgM is an antibody that can be secreted by the mucosa, including in the upper airways, why IgM levels were not evaluated? If was measured, it could be interesting to report these data.

- Thank you for your valuable comment. We understand your interest in mucosal IgM class antibodies. However, Waldman et al. detected IgG and IgA, but not IgM, in bronchoalveolar lavage fluids and nasal washings after mucosal immunization of inactivated IAV vaccine (Ref. [25]). Also, in a common-cold coronavirus infection, it was shown that IgG and IgA, but not IgM, can persist for extended periods in the serum and nasal fluids (Ref. [26]). Therefore, we focused on IgA and IgG antibodies in saliva and did not measure the IgM antibody. We have added this statement in the last paragraph of the introduction.

4) Please state what was the volunteer groups enrolled in this study since was not clear the reason to cite vaccination for the Influenza virus or why the authors reported a result concerning one asymptomatic individual.

- Thank you for your comment.

- For sampling, the volunteers have been recruited in the department of medical technology where the first author belongs. Because these students have to learn how to take blood from each other and use their serum for several clinical tests as training, their serum samples were stocked every year. When they practice in the hospital at some period, they are requested to have an influenza vaccine. Taking this advantage, we planned to collect saliva and serum samples on vaccination and on infection, which was approved by the ethical committee. Since 2017, the students who got influenza have been called for cooperation. The year 2019/2020 season was a rare occasion that many students whose saliva samples were stocked were infected with H1N1 influenza. Furthermore, we had an opportunity to know the situation of close contact infection in detail among 3 students. We believe that this study could only be done in this special situation and has led us to important and suggestive findings.

- I added the following sentence in the Acknowledgments; The sampling and antibody measuring was done partly by the active cooperation of those students who learn technologies for clinical laboratories.

In the Introduction section:

5) The authors declare that nasal and saliva could be useful to evaluate the antibodies levels in the upper airways. However, it is noteworthy to mention that, whereas secretory IgA (SIgA), SIgM, and IgG can be detected in saliva samples, IgG is not easily detected in nasal fluids. Therefore, it is recommended to highlight this fact in the "Introduction" section in order to reinforce the use of saliva to monitor these antibodies responses in the study context.

- Thank you for your valuable comment. I did not know that IgG is not easily detected in nasal fluids. As I described in the response of 3), in a common-cold coronavirus infection, IgG and IgA responses persisted for extended periods in the serum and nasal fluids (Ref. no. 26]). Also, please note that in the nasal vaccine studies, both HA-specific IgG and IgA were detectable in the nasal wash, though the level of IgG is lower than that of IgA (Ref. no. 12, 13). Because the extensive concentration of a large amount of nasal wash (~100 ml) was required for measuring these antibody titers, the saliva sampling has great advantage as I described in the introduction and also in discussion.

In the Material and Methods section:

6) Please state that the study followed the Helsinki declaration and also it is necessary to present the study approved number from Ethics Committee.

- The approved number from the ethical committee is given on page 9 of the revised manuscript.

7) How the serum was obtained?

- As we commented in 4), blood sampling is a routine task as a medical technologist. In this study, 2~3 ml blood was taken either by a medical doctor or students under the supervision of a medical doctor in the university.

8) Please clarify whether the samples used in the group vaccinated were obtained in a totally different group from the influenza-infected group

- Yes, the saliva samples of the group vaccinated are collected and stocked during the 2018/2019 flu season (Table 2). It is completely different individuals from the influenza-infected in 2019/2020 flu season (Talbe1).

- We added a phrase on page 8.

9) Since some analyses were performed with samples obtained on three different occasions, Friedman's test with Dunn's post hoc test should be used.

- Thank you for your comment. You may have meant “three different occasions” as pre, early, and late time points in Fig. 2. However, individuals have variable basal antibody titer before IAV infection. So, basically, the pre titer needs to be used to assess the increased level of antibody in virus infection, which is a “fold increase” as depicted in the right panel. Because Friedman’s test is a nonparametric test that compares three or more paired groups, we did not apply it here.

In the Results section

10) In order to be clearer, I suggest reorganizing the following sentences on page 12, lines 185-188, as described below.

"Notably, donor nos. 2003, 2006, 2007, and 2016 were never vaccinated. In contrast, donor nos. 2005, 2008, and 2013 were vaccinated every year, including the 2019–2020 flu season. Concerning the HI titer against the A(H3N2) influenza virus, it was found <40 in all the serum samples."

- Thank you for your helpful suggestion. We amended this part according to your suggestion.

11) The authors did not report that the specific IgG levels for HIHA from the volunteer 2006 were increased in time point "pre" (Fig 1b). This is an interesting result since the HI titer of this volunteer was under the detection rate at the same time point (Fig. 1a).

- The reviewer may point out the relatively high level of HA-specific IgG before IAV infection in no. 2016 (not 2006) serum with no HI activity. We added the following comment on page 13; “Because the “pre” serum was available only in no. 2016 and no. 2013, it is not clear whether the relatively high basal level of HA-specific serum IgG in no. 2016 is within the variability of ELISA measurement or due to some other reasons.”

12) Please remove the last sentence on page 12, lines 195-196, and the first paragraph of page 13, lines 197-200, due to the fact that these pieces of information were already presented in the "introduction" section.

- We completely agree with the reviewer. We removed the first paragraph.

13) Although the suggestion presented on page 13, lines 209-211, are relevant, I believe that these pieces of information could be useful in to "Discussion" section. So, please remove it from the "Results" section to the "Discussion" section.

- Thank you for your suggestion. However, in many pieces of literature, the brief summary comment at the end of the section is helpful for readers to understand the contents and author’s claim. We prefer to leave the last sentence as it is.

14) In Fig. 3a it is possible to observe that the values of A(H1N1)pdm09-specific salivary IgA from the volunteer V11 were increased post-vaccination. Corroborating this observation the authors cited, on page 14, lines 217-218, that "...the IgA levels remained largely unchanged after vaccination except in V11." So, I would like to know whether the values obtained pré and post-vaccination were significantly different?

- We are sorry to confuse you. The values obtained pre and post-vaccination were not statistically different. We added the following sentence on page 15; Thus, basically, the sIgA was not increased by vaccination.

-

15) Are there statistical differences in the results presented in Figs. 4a and 4b?

- Thank you for the comment. The results are findings of serum and salivary antibodies only in three individuals. We think that the statistical analysis is not applicable here.

16) Again, on page 14, lines 227-230, as well as on page 15, lines 242-247, the authors presented a putative conclusion or even suggestions for the results found. I recommend removing these sentences since this section must only present the results obtained in the study.

- We agree with the reviewer’s suggestion here. We removed these sentences to the discussion.

In the Discussion section:

17) The authors discuss, in a very well way, the potential use of saliva for monitoring not only the influenza virus infection but also its annual vaccination. However, some points could be improved:

Although saliva could be considered a "corollary" fluid to evaluate the mucosal immunity of upper airways, there are some specific differences in oral and nasal mucosa immunity that must be discussed. Therefore, I suggest reorganizing some pieces of information from the "Introduction" section to the "Discussion" section.

In addition, the "time" of mucosal immunity response should be more discussed since this fact could impact the evaluations presented in this study. For instance, as recently mentioned by Dos Santos et al.*, "...the literature demonstrates that SIgA is an important tool for early detection of infections, normally, 1 day after the infection, whereas serum IgA and IgM can be detected after 3–5 days after the infection (30)".

* Dos Santos JMB, Soares CP, Monteiro FR, Mello R, do Amaral JB, Aguiar AS, Soledade MP, Sucupira C, De Paulis M, Andrade JB, Almeida FJ, Sáfadi MAP, Mau LB, Brasil JM, Ramalho T, Loures FV, Vieira RP, Durigon EL, de Oliveira DBL, Bachi ALL. In Nasal Mucosal Secretions, Distinct IFN and IgA Responses Are Found in Severe and Mild SARS-CoV-2 Infection. Front Immunol. 2021 Feb 25;12:595343. doi: 10.3389/fimmu.2021.595343. eCollection 2021.

- Thank you for your helpful suggestions. We thoroughly re-organized the discussion. We hope you will find the resolution of your concerns in the new discussion section.

- With respect to the time of mucosal response, the reference you have cited is the Dengue virus infection, not upper respiratory. Instead, I added the following in the discussion on page 20 (marked yellow); In this context, a wave of IgA plasma cells in the blood is considered to occur prior to the production of secretory IgA antibodies with a peak at around 6-10 days after mucosal infection/immunization (ref. No.24). As the reviewer #2 pointed out, the much earlier time course of antibody response in saliva would be interesting to be studied further in nasal vaccinations.

Submitted filename: Yokota Response to Reviewers.docx

Click here for additional data file.

19 Jan 2022

Saliva as a useful tool for evaluating upper mucosal antibody response to influenza

PONE-D-21-28281R1

Dear Dr. Tsunetsugu-Yokota,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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,

Paulo Lee Ho, Ph.D.

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 #2: 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 #2: Yes

**********

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

Reviewer #2: 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

**********

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

**********

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: (No Response)

**********

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: André LL Bachi

28 Jan 2022

PONE-D-21-28281R1

Saliva as a useful tool for evaluating upper mucosal antibody response to influenza

Dear Dr. Tsunetsugu-Yokota:

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

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Paulo Lee Ho

Academic Editor

PLOS ONE