Mucosal response of inactivated and recombinant COVID‐19 vaccines in Congolese individuals

Abstract Background The efficacy of immunization against an airborne pathogen depends in part on its ability to induce antibodies at the major entry site of the virus, the mucosa. Recent studies have revealed that mucosal immunity is poorly activated after vaccination with messenger RNA vaccines, thus failing in blocking virus acquisition upon its site of initial exposure. Little information is available about the induction of mucosal immunity by inactivated and recombinant coronavirus disease 2019 (COVID‐19) vaccines. This study aims to investigate this topic. Methods Saliva and plasma samples from 440 healthy Congolese were collected including (1) fully vaccinated 2 month postvaccination with either an inactivated or a recombinant COVID‐19 vaccine and (2) nonvaccinated control group. Total anti‐severe acute respiratory syndrome coronavirus 2 receptor‐binding domain IgG and IgA antibodies were assessed using in‐house enzyme‐linked immunosorbent assays for both specimens. Findings Altogether, the positivity of IgG was significantly higher in plasma than in saliva samples both in vaccinated and nonvaccinated control groups. Inversely, IgA positivity was slightly higher in saliva than in plasma of vaccinated group. The overall IgG and IgA levels were respectively over 103 and 14 times lower in saliva than in plasma samples. We found a strong positive correlation between IgG in saliva and plasma also between IgA in both specimens (r = .70 for IgG and r = .52 for IgA). Interestingly, contrary to IgG, the level of salivary IgA was not different between seropositive control group and seropositive vaccinated group. No significant difference was observed between recombinant and inactivated COVID‐19 vaccines in total IgG and IgA antibody concentration release 2 months postvaccination both in plasma and saliva. Conclusion Inactivated and recombinant COVID‐19 vaccines in use in the Republic of Congo poorly activated mucosal IgA‐mediated antibody response 2 months postvaccination.


| INTRODUCTION
The worldwide impact of the coronavirus disease 2019 (COVID-19) pandemic has called for the rapid development of public health measures to control the spread of the virus. 1 In some ways, however, the virus is under better control since the first cases were identified in Wuhan, China, on December 2019 due to different fighting deployed measures including vaccination. 2 Regardless of the benefits of those COVID-19 vaccines in curbing the pandemic, there are still some concerns about the patterns underlying the protection conferred by anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies following vaccination, as vaccinated individuals continue to be infected.][5][6] Current evidence suggests that mucosal immunity plays an important role in protecting against most respiratory infections including influenza, 7 poliovirus, 8 and SARS-CoV-2, 6,9 as it involves the activation of immune cells and secretion of antibodies directed towards mucosal tissues of the respiratory tract, which, in case of SARS-COV-2, can help to prevent the virus spike protein to fix in the ACE2 receptor and invade host cells. 6ecretory immunoglobins A (sIgA) are the most abundant antibody isotype found in mucosal secretions, such as saliva and mucus.It is produced by plasma cells located in the mucosal-associated lymphoid tissue and is transported across mucosal surfaces to impede viral replication and shedding in the airways. 6,10]14 Recombinant vaccines (Sputnik/rAd26, Janssen/ Ad26.COV2.S) and inactivated vaccines (Sinopharm/ BBIP-CorV) are the most distributed COVID-19 vaccines so far (SITREP 208, SITREP 244) in the Republic of Congo (RoC).6][17][18][19] However, little is known about the mucosal immunity induced by the aforementioned vaccines.The objective of the current study is to assess anti-receptor-binding domain (RBD) antibody IgG and IgA in the saliva and plasma following Sinopharm/BBIP-CorV, Janssen/Ad26.-COV2.S, and Sputnik/rAd26 vaccination in Congolese participants.

| Study design and participants
The study has been conducted from May to August 2022 in Brazzaville (capital of the RoC), to assess both prevalence and level of mucosal and serum antibodies in the population.A call for participation in the study was launched and interested people aged more than 18 years old were welcomed at the health facility of the Congolese foundation for medical research (Brazzaville) for enrollment if they meet the inclusion criteria.Informed consent was given before enrollment and after clinical examination, the clinician recorded sociodemographic and clinical data and vaccine description (brand, type, date of vaccination).The study clinical protocol was approved by the Institutional Ethics Committee of the Congolese Foundation for Medical Research (038/CIE/FCRM/2022).

| Treatment of samples
Oropharyngeal swabs was performed for SARS-CoV-2 infection screening by PCR using the QIAamp Viral RNA Mini Kit (Qiagen) for RNA extraction and subjected to RealStar® SARS-CoV-2 (Altona Diagnostics) for RNA detection.Positive individuals were excluded from the final analysis.
From each negative participant, blood (5 mL) was collected using lithium heparin monovets or EDTA and obtained plasma was stored at −20°C.Saliva was collected by spitting into a simple plastic tube (multipurpose containers 50 mL Greiner Bio-One ref. 201150).Before sample collection, participants were asked to refrain from consuming food, drink, or tobacco products for at least 30 min.Each enrolled subject underwent self-collection of whole saliva samples under the supervision of a trained healthcare professional.Participants were instructed not to swallow saliva for at least 30 s to 1 min, to accumulate a significant amount before spitting into a sterile container.Saliva samples were immediately transferred in two (2) 1.5 or 2 mL reaction tubes and kept at −20°C.Twenty-five additional plasma samples from archived prepandemic period (before 2019) were used as negative control.

| Measurement of antigen-specific SARS-CoV-2 IgG in saliva and plasma
In-house enzyme-linked immunosorbent assays (ELI-SAs) specifically established to analyze IgG antibodies in plasma and saliva, reacting against the SARS-CoV-2 RBD of the ancestral strain were used. 20Briefly, high-binding plates (Corning, ref: 3590) were coated overnight with 50 µl per well of 2 µg/mL of the SARS-CoV-2 RBD antigen suspended 1× phosphate-buffered saline (PBS) (Gibco, ref: 18912-014).Wells were washed once with 1× PBS without detergent, 200 µL per well, and blocked with 100 µL of The Blocking Solution (Candor Bioscience GmbH) for 2 h at room temperature (RT) on a microplate shaker (700 rpm).Plasma samples of participants and 25 archived prepandemic plasma were serially diluted from 1:100 to 1:62,500 using the blocking solution.The plates were washed three times with 200 µL per well of × PBS + 0.1% Tween 20.Then, 100 mL of sample dilution was added per well and incubated for 1 h (RT, 700 rpm).After plate washing, 50 µL per well of an horseradish peroxidase (HRP)-coupled antihuman IgG was used (Jackson Immuno Research Laboratories, ref. 109-036-097).The detection antibody was diluted 1:10.000 in 1× ROTI Block buffer (Carl Roth, ref.A151.2) and incubated for 30 min (RT, 700 rpm).For visualization, 100 µL TMB substrate solution was added after four times washing and the reaction was stopped using 1 M HCl.Plates were measured at 450 nm and 620 nm with a microplate reader (CLARIOstar, BMG LABTECH).The IgG concentration was presented in µg/mL and the cut-off value was previously set to 4.0 µg/mL. 20The SARS-CoV-2 IgG detection in saliva was performed following similar parameters as used in the plasma ELISA assay, with some exceptions: Saliva was diluted from 1:3 up to 243 using the same blocking solution.For IgG detection, a 1:20,000 in 1× ROTI Block diluted biotinylated antihuman IgG was incubated for 1 h and 1:1000 Avidin-HRP (Biolegend ref. 405103) was applied for 30 min.The cutoff for salivary SARS-CoV-2 IgG positivity was previously set to 6.3 ng/mL. 20

| Measurement of antigen-specific SARS-CoV-2 IgA in saliva and plasma
An in-house ELISA specifically established to analyze IgA in plasma and saliva reacting to SARS-CoV-2 RBD of the ancestral strain were used. 20The same parameters for IgG were also used here with some exceptions: High binding plates were coated overnight with 50 µL per well of 1 µg/mL of the SARS-CoV-2 RBD antigen for IgA in plasma and 2 µg/mL in saliva.Wells were blocked with 200 mL of The Blocking Solution (Candor) for IgA plasma and 10% of milk prepared in 1× PBS with 0.1% Tween 20 for saliva (incubated for 2 h at RT). Plasma samples and 25 archived prepandemic plasma were serially diluted from 1:100 to 1:12,500 using the blocking solution.For Plasma IgA detection, an HRP-coupled antihuman IgA was used (Jackson LOT#109-035-011).The detection antibody was diluted 1:5000 in 1× ROTI Block buffer and incubated for 30 min (RT, 700 rpm).The cutoff for plasma SARS-CoV-2 IgA positivity was previously set to 0.5 µg/mL. 20Saliva samples and controls were serially diluted from 1:3 to up to 1:81 using the 10% milk and for salivary IgA detection a 1:10,000 diluted biotinylated antihuman IgA (in milk) and 1:1000 Avidin-HRP in 1× Roti were used.
A cutoff for IgG in plasma/saliva and IgA in plasma was already available as described by. 20We have confirmed their effectiveness by using pre-pandemic samples and finding all samples being negatives for IgA (0 positive out of the 25 samples) and one sample (1/25) unfortunately barely crossing the cut-off line for IgG (Figures 4B and 6B).

| Statistical analysis
RStudio (Version 1.2.5001), running R (version 4.2.1.),and Graph Pad Prism (Version 8.0.2) were used for statistical analyses.The significance was defined by a 95% confidence interval (p < .05).Categorical variables were reported as percentages and compared using Fisher's exact test.Continuous variables were reported as median (interquartile range, IQR).The difference in antibody levels between two groups was analyzed with Mann-Whitney test, whereas the Kruskal-Wallis test followed by Dunn's multiple comparison test was used for comparing more than two groups.Correlations between antibody concentrations were performed using Pearson's test.

| Characteristics of the participants
A total of 440 participants were recruited including 193 vaccinated and 247 nonvaccinated control group (Figure 2).Males were significantly more represented than female (Fisher's exact test: p = .0160).The median age of the participants was 27 years (ranging from 18 to 83).The demographic characteristics of the included participants are shown is the Table 1.

| Total anti-SARS-COV-2 IgG positivity in plasma and saliva samples in vaccinated participants
The overall result revealed that the estimated IgG positivity in non-vaccinated control group was significantly higher in plasma (211 positive out of the 244; 86.4%) than in saliva (151 positive out of the 237; 64%; Fisher's exact test: p = .0003).The same was observed in vaccinated group with 96.9% (186/192) of the positivity in plasma versus 94.2% (179/190) in saliva but no significantly p = .5958(Figure 3).

| Total anti-SARS-COV-2 IgG level in plasma and saliva samples in vaccinated participants
We assessed and compared the magnitude of the total IgG response of seropositive (SP) individuals according to the specimen and groups.The results revealed that the median IgG concentration was over 1000-fold higher in plasma than in saliva of the SP nonvaccinated (control group, SP) p = .0001.The same was observed in SP vaccinated group (vaccinated, SP) p = .0001(Figure 4A).When comparing IgG antibody level between saliva of vaccinated, SP, and saliva of the control group, SP results showed that vaccinated individuals harbored significantly higher IgG level than control group p = .0001.The same was found in plasma (Figure 4B,C and Table 2).Correlations analysis further indicated that the level of salivary IgG of the participants positively correlated with that of plasma IgG antibody (r = .70,p = .0001)(Figure 4D).

| Total anti-SARS-COV-2 IgA positivity in plasma and saliva samples in vaccinated participants
The estimated IgA positivity in nonvaccinated control group was higher in plasma (72 positive out of the 244; 29.5%) than in saliva with no significance (51 positive out of the 205; 24.8%; Fisher's exact test: p = .5267).However, not the same was observed in vaccinated group where we found IgA positivity being slightly higher in the saliva (127 positive out of the 186; 68.2%) than in the plasma (105 positive out of the 192; 54.6%; Fisher's exact test: p = .809)with no significance (Figure 5).

| Total anti-SARS-COV-2 IgA levels in plasma and saliva samples in vaccinated participants
When assessing and comparing the magnitude of the total IgA response of SP individuals according to the  specimen and group, the results revealed that the median IgA level was about over 14-fold higher in plasma than in saliva of the SP nonvaccinated group (control group, SP) p = .0001.The same was observed in SP vaccinated group (vaccinated, SP) p = .0001(Figure 6A).We found the similar IgA antibody level between saliva of vaccinated and saliva of the control group p = .5599.The same observation was made in plasma sample p = .8673(Figure 6B,C and Table 2).Correlation analysis further indicated that the level of salivary IgA of the participants positively correlated with that of plasma (r = .52,p = .0001)(Figure 6D).

| Assessment of total IgA and IgG level in saliva and plasma according to the type of vaccine
When evaluating antibody response according to the type of the vaccine received, results indicate that the three vaccines (Ad26.COV2.S, rAd26, and BBIP-CorV vaccines) induced IgG and IgA level with no differences both in plasma and saliva (Figure 7 and Table 3).

| DISCUSSION
Understanding the magnitude and compartmentalization of antibody in a diverse population is essential in designing mass vaccination strategies taking into account the type of vaccine to use and its effectiveness.We evaluated the magnitude of salivary antibody release after vaccination with either an inactivated (BBIP-CorV) or a recombinant (rAd26 and Ad26.COV2.S) COVID-19 vaccine in use in the RoC during the study period in comparison with nonvaccinated control group.We found that SARS-CoV-2 IgG positivity was higher in plasma than in saliva in both vaccinated and nonvaccinated control group.This could be explained by the fact that most of the salivary IgG antibodies likely originate in the plasma by transudation from the bloodstream. 21To further clarify this point, we assessed the magnitude of the total anti-SARS-CoV-2 IgG antibodies in saliva and plasma then processed multiple comparisons and correlation analyses.We found a median concentration of IgG approximately a 1000 times higher in plasma than in the saliva.We also found a high positive correlation between salivary IgG and plasma IgG, supporting the hypothesis of a plasma derived origin of salivary IgG. 21,22When comparing the IgG antibody magnitude in saliva samples of positive groups (i.e., saliva of positive vaccinated vs. saliva of positive control group), we found vaccinated displaying a significantly higher level of IgG than of the control group.The same was observed in plasma.An explanation could be the potent immunogenic properties of the administered COVID-19 vaccines, as previously reported. 15,16,18,23urthermore, the robustness of the IgG antibody response in naturally exposed (here positive control group) individuals has been reported to vary depending on the severity of symptoms (asymptomatic, mild symptomatic, and highly symptomatic). 24n contrast to IgG, we found that the overall IgA positivity was slightly higher in saliva than in plasma of vaccinated.At first sight, this means that vaccination has induced IgA released at plasma and saliva site.6][27][28] Together, this could explain why the IgA positivity was a bit higher in saliva than in plasma.To further bring light in this point, we assessed the magnitude of the total anti-SARS-CoV-2 IgA in saliva and plasma of the overall participants then processed multiple comparisons and correlation analysis.We found a median concentration of IgA ~14-fold higher in plasma of participants than in saliva.We also found a high positive correlation between salivary IgA and plasma IgA (r = .52).These findings suggest that the majority of salivary IgA evidenced here was a plasma-derived isotype (mIgA). 21urther analysis would better confirm this.When comparing the IgA antibody magnitude in saliva samples of positive groups (i.e., saliva of positive F I G U R E 5 Seroprevalence of anti-severe acute respiratory syndrome coronavirus 2 IgA in plasma and saliva by vaccinated group and nonvaccinated control group.Fisher's exact test was used for the comparison of IgA seroprevalence between saliva and plasma among groups.vaccinated vs. saliva of positive control group), interestingly, we found the similar IgA antibody level between both groups.The same was observed in plasma.This result suggest that the COVID-19 vaccines used in the RoC during the study period would not have induced a robust mucosal response associated with IgA.Similar results were recently reported for people vaccinated with mRNA COVID-19 vaccines, [3][4][5]29 indicating that COVID-19 vaccines in general do not induce a robust mucosal IgA associated antibody response, which may explain the reinfection seen worldwide among vaccinated. 30The analysis of the total amount of IgG and IgA released after vaccination with Recombinant vaccines (Sputnik/rAd26, Janssen/Ad26.COV2.S) and inactivated vaccine (Sinopharm/BBIP-CorV), both in saliva and plasma samples, show no difference, indicating that all those vaccines were equally immunogenic.
The overall high IgG prevalence in non-vaccinated control group in this study indicates that the population of Brazzaville, the epicenter of the pandemic in the RoC, already achieved herd immunity against SARS-COV-2.This is supported by a low PCR positivity rate reported in the national situation report (SITREP) during the study period (turning around 2%) [SITREP 208-244] and, also by considering our previous findings. 31,32Nevertheless, the state of herd immunity is probable to be bypassed by other viral variants, which are less efficiently blocked by the antibodies generated in previous infections.

F 1 F 4
I G U R E 2 Recruitment of participants.T A B L E 1 Characteristics of study population.I G U R E 3 Seroprevalence of anti-severe acute respiratory syndrome coronavirus 2 IgG in plasma and saliva by vaccinated group and nonvaccinated control group.Fisher's exact test was used for the comparison of IgG seroprevalence between saliva and plasma among groups.Level of severe acute respiratory syndrome coronavirus 2-specific total IgG in plasma and saliva of seropositives (SPs).(A) Level of total IgG antibody in plasma versus saliva specimens of vaccinated and control group.(B) Level of total IgG in plasma of SP vaccinated group (n = 186) versus of nonvaccinated SP group (n = 211) and prepandemic (n = 25).(C) Level of total IgG in saliva of SP vaccinated (n = 179) versus nonvaccinated SP group (n = 151).(D) IgG Pearson correlation between saliva and plasma.The horizontal lines show the median values, the box plots show the interquartile range and the whiskers indicate the minimum-to-maximum range.Each dot corresponds to an individual subject.p values were determined using the Mann-Whitney.T A B L E 2 Summary of the relevant comparison on the magnitude of the total IgG and IgA response in plasma and saliva of positive (SP) participants.

6
Level of severe acute respiratory syndrome coronavirus 2-specific total IgA in plasma and saliva of seropositive (SP).(A) Level of total IgA antibody in plasma versus saliva specimens of vaccinated and control group.(B) Level of total IgA in plasma of SP vaccinated group (n = 105) versus of nonvaccinated SP group (n = 72) and prepandemic (n = 17).(C) Level of total IgA in saliva of SP vaccinated (n = 127) versus nonvaccinated SP group (n = 51).(D) IgA Pearson correlation between saliva and plasma.The horizontal lines show the median values, the box plots show the interquartile range and the whiskers indicate the minimum-to-maximum range.Each dot corresponds to an individual subject.p values were determined using the Mann-Whitney.
33,345 | CONCLUSIONInactivated and recombinant COVID-19 vaccines in use in the RoC would not have induced a robust mucosal response associated with IgA.Median IgG and IgA severe acute respiratory syndrome coronavirus 2 antibody concentration release in plasma and saliva after vaccination with Ad26.COV2.S, rAd26, and BBIP-CorV vaccines.The horizontal lines show the median values, each dot corresponds to an individual subject.p values were determined using the Kruskal-Wallis test with the Dunn's multiple comparison test.Comparison of the median IgG and IgA SARS-CoV-2 antibody release between Sputnik/rAd26, Janssen/Ad26.COV2.S, and Sinopharm/BBIP-CorV COVID-19 vaccine.: COVID-19, coronavirus disease 2019; IQR, interquartile range; SARS-Cov-2, severe acute respiratory syndrome coronavirus 2.
F G U R E 7 T A B L E 3Abbreviations