Natural SARS-CoV-2 Infection Affects Neutralizing Activity in Saliva of Vaccinees

Abstract

Background: SARS-CoV-2 transmission mainly occurs through exposure of the upper airway mucosa to infected secretions such as saliva, which are excreted by an infected person. Thus, oral mucosal immunity plays a central role in the prevention of and early defense against SARS-CoV-2 infection. Although virus-specific antibody response has been extensively investigated in blood samples of SARS-CoV-2-infected patients and vaccinees, local humoral immunity in the oral cavity and its relationship to systemic antibody levels needs to be further addressed.

Material and methods: We fine-tuned a virus neutralization assay (vNTA) to measure the neutralizing activity (NA) of plasma and saliva samples from 20 SARS-CoV-2-infected (SI), 40 SARS-CoV-2-vaccinated (SV), and 28 SARS-CoV-2-vaccinated subjects with a history of infection (SIV) using the "wild type" SARS-CoV-2 lineage B.1 (EU) and the Delta (B.1.617.2) strains. To validate the vNTA results, the presence of neutralizing antibodies (NAbs) to the spike receptor binding domain (RBD) was evaluated with an ELISA assay.

Results: NA to SARS-CoV-2 lineage B.1 (EU) was present in plasma samples from all the tested subjects, with higher titers in SIV compared to both SI and SV. Conversely, NA was detected in saliva samples from 10.3% SV, 45% SI, and 92.6% SIV, with significantly lower titers in SV compared to both SI and SIV. The detection of NAbs in saliva reflected its reduced NA in SV.

Discussion: The difference in NA of plasma vs. saliva was confirmed in a vNTA where the SARS-CoV-2 B.1 and Delta strains were tested head-to-head, which also revealed a reduced NA of both specimens compared to the B.1 variant.

Conclusions: The administration of SARS-CoV-2 vaccines was associated with limited virus NA in the oral cavity, as measured in saliva and in comparison to plasma. This difference was more evident in vaccinees without a history of SARS-CoV-2 infection, possibly highlighting the importance of local exposure at the site of virus acquisition to effectively prevent the infection and block its spread. Nevertheless, the presence of immune escape mutations as possibly represented by the SARS-CoV-2 Delta variant negatively affects both local and systemic efficacy of NA associated with vaccination.

Keywords: SARS-CoV-2; antibodies; neutralizing activity; saliva; variants.

Figure 1

Graphical representation of the study workflow. SARS-CoV-2-infected (SI), vaccinated (SV), and infected–vaccinated (SIV) subjects were enrolled in the study (Step 1). Blood and saliva samples were collected and processed (Step 2) so as to be analyzed for neutralizing activity (NA) by SARS-CoV-2 neutralization assay (NTA), neutralizing antibodies (Nabs) by enzyme-linked immunosorbent assay (ELISA), as well as cytokine production (Multiplex ELISA).

Figure 2

Neutralizing activity (NA) from plasma and saliva of SARS-CoV-2-infected and/or vaccinated subjects, measured by virus neutralization assay (vNTA). NA in plasma and saliva samples are reported in panel (A). Correlation between NA in plasma and saliva samples of all tested subjects are showed in panel (B), while correlation between NA in plasma and saliva specimens of SARS-CoV-2-infected (SI), SARS-CoV-2-infected and vaccinated (SIV), and SARS-CoV-2-vaccinated (SV) subjects are represented in panels (C–E), respectively. ***p < 0.0001.

Figure 3

Neutralizing activity (NA) and anti-RBD NAbs titer in saliva samples from SARS-CoV-2-infected and/or vaccinated subjects. (B) NA quantified by vNTA in a subgroup of SV (n = 18) and SIV (n = 15) subjects is reported in panel (A). In the same subgroup, anti-RBD NAb production was detected by ELISA assay. The dashed line is representative of a cutoff equal to 20%. *p < 0.05, ***p < 0.001. (C) Taking into account all the subjects (SV+SIV), we observed a positive correlation between salivary NA tested by the two techniques (vNTA and ELISA). The production of anti-RBD NAbs quantified by ELISA was positively correlated to the NA tested by NTA in SIV (D) but not in the SV group (E).

Figure 4

vNTA against SARS-CoV-2 lineage EU (B.1) and Delta variant (B.1.617.2). Virus neutralization assay (vNTA) titer on the Delta variant was significantly lower compared to the «wild type» SARS-CoV-2 (EU) in both plasma (A) and saliva (B) samples. *p < 0.05. Comparison between the EU variant and Delta variant in plasma and saliva samples from each enrolled subject is reported in panels (C, D), respectively. Lines connect the NAbs of each individual subject. In panels (E) (plasma) and (F) (saliva), vNTA correlation between the EU and the Delta variant is described.