Molecular Events in Immune Responses to Sublingual Influenza Vaccine with Hemagglutinin Antigen and Poly(I:C) Adjuvant in Nonhuman Primates, Cynomolgus Macaques

Abstract

Sublingual vaccines offer the benefits of inducing mucosal immunity to protect against respiratory viruses, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and influenza, while also enabling needle-free self-administration. In a previous study, a sublingual SARS-CoV-2 vaccination was created by combining a recombinafigureCoV-2 spike protein receptor-binding domain antigen with a double strand RNA Poly(I:C) adjuvant. This vaccine was tested on nonhuman primates, Cynomolgus macaques. This study examined the immune and inflammatory responses elicited by the sublingual influenza vaccine containing hemagglutinin (HA) antigen and Poly(I:C) adjuvants, and assessed the safety of this vaccine in nonhuman primates. The Poly(I:C)-adjuvanted sublingual vaccine induced both mucosal and systemic immunities. Specifically, the sublingual vaccine produced HA-specific secretory IgA antibodies in saliva and nasal washings, and HA-specific IgA and IgG were detected in the blood. This vaccine appeared to be safe, as judged from the results of blood tests and plasma C-reactive protein levels. Notably, sublingual vaccination neither increased the production of inflammation-associated cytokines-IFN-alpha, IFN-gamma, and IL-17-in the blood, nor upregulated the gene expression of proinflammatory cytokines-IL12A, IL12B, IFNA1, IFNB1, CD69, and granzyme B-in white blood cells. Moreover, DNA microarray analyses revealed that sublingual vaccination evoked both enhancing and suppressing expression changes in genes associated with immune-related responses in cynomolgus monkeys. Therefore, the sublingual vaccine with the Poly(I:C) adjuvant is safe, and creates a balanced state of enhancing and suppressing the immune-related response.

Keywords: CLEC4G; LSECtin; efficacy; monkey; prophylaxis; tolerance.

Figure 1

Points in time for sublingual vaccinations and assessments of anti-HA (hemagglutinin) antibodies, blood tests, plasma cytokines, quantitative reverse transcription PCR (RT-qPCR), and DNA microarray analyses. RT-qPCR and DNA microarray analyses were conducted with RNA isolated from white blood cells (WBC). Vaccinations were performed four times at 0 (1st), 6 (2nd), 18 (3rd), and 30 (4th) weeks. Arrows indicate sampling timepoints for each assay.

Figure 2

HA-specific antibodies induced using the sublingual vaccination with HA + Poly(I:C). (A) Anti-HA sIgA in saliva, (B) Anti-HA sIgA in nasal washings, (C) Anti-HA IgA in plasma, and (D) Anti-HA IgG in plasma. Open circles () indicate the control group, and solid brown circles () indicate the experiment/HA + Poly(I:C)) group. Red arrows () indicate vaccination. Relative titers were estimated using the conditions mentioned in Section 2.

Figure 3

Cytokine production in the experiment (HA + Poly(I:C)) group at the commencement of vaccination (W0:0), and 1 week after the third vaccination (W19:19). (A) IFN-alpha, (B) IFN-gamma, and (C) IL-17. The levels of cytokines were expressed as pg/mL of plasma.

Figure 4

Gene expression (mRNA) levels of cytokines and effector molecules in the experiment (HA + Poly(I:C)) group at the time points: pre-vaccination (W0:0), and 1 week after (W19:19) the third vaccination. (A) IL12A, (B) IL12B, (C) IFNA1, (D) IFNB1, (E) CD69, and (F) GZMB. Red arrows indicate vaccination.

Figure 5

Balanced immune response elicited by the sublingual vaccine with an HA antigen and Poly(I:C) adjuvant.