Evaluation of mucosal adjuvants and immunization routes for the induction of systemic and mucosal humoral immune responses in macaques

Abstract

Delivering vaccine antigens to mucosal surfaces is potentially very attractive, especially as protection from mucosal infections may be mediated by local immune responses. However, to date mucosal immunization has had limited successes, with issues of both safety and poor immunogenicity. One approach to improve immunogenicity is to develop adjuvants that are effective and safe at mucosal surfaces. Differences in immune responses between mice and men have overstated the value of some experimental adjuvants which have subsequently performed poorly in the clinic. Due to their closer similarity, non-human primates can provide a more accurate picture of adjuvant performance. In this study we immunised rhesus macaques (Macaca mulatta) using a unique matrix experimental design that maximised the number of adjuvants screened while reducing the animal usage. Macaques were immunised by the intranasal, sublingual and intrarectal routes with the model protein antigens keyhole limpet haemocyanin (KLH), β-galactosidase (β-Gal) and ovalbumin (OVA) in combination with the experimental adjuvants Poly(I:C), Pam3CSK4, chitosan, Thymic Stromal Lymphopoietin (TSLP), MPLA and R848 (Resiquimod). Of the routes used, only intranasal immunization with KLH and R848 induced a detectable antibody response. When compared to intramuscular immunization, intranasal administration gave slightly lower levels of antigen specific antibody in the plasma, but enhanced local responses. Following intranasal delivery of R848, we observed a mildly inflammatory response, but no difference to the control. From this we conclude that R848 is able to boost antibody responses to mucosally delivered antigen, without causing excess local inflammation.

Keywords: Macaque; Mucosal; TLR ligand; adjuvant; inflammation; intranasal.

Figure 1.

R848 is a potent mucosal adjuvant. Macaques were immunised intranasally with 200 μg KLH in combination with 500 μg of the adjuvants R848, Pam3SCK4 or chitosan, or 50 μg of TSLP or in PBS alone (n = 4 per group). Immunizations were administered at weeks 0, 4 and 8. KLH specific IgG was measured in plasma for all groups (A). Further analysis was performed for animals IE73, IE74, GK02 and IF53 from the R848/ KLH group and IE71 from the TSLP group. KLH specific ELISA were performed for IgG (C, E) and IgA (B, D, F) on plasma (B), nasal wash (C, D) and vaginal Weck-cels (E, F). KLH specific scIgA was measured in nasal samples (G). Relative coefficients of excretion (RCE) compared to albumin in nasal and vaginal samples (H). Data is presented as mean +/− SD of n = 4 animals (A, H) or individual animals (B-G).

Figure 2.

R848 induces antibody responses after mucosal or systemic immunization. Macaques were immunised intramuscularly (IM, open symbols, A, B) or intranasally (IN, closed symbols, C, D) with 200 μg KLH in combination with 500 μg R848 (n = 4 per group). Immunizations were administered at weeks 0, 4 and 8. KLH specific ELISA were performed for IgG (A, C) and IgA (B, D) in plasma, data is presented as individual animals. Pooled data for each route is presented for IgG (E) and IgA (F), where each point represents mean of the n = 4 animals presented in A-D. * p < 0.05, ** p < 0.01 by multiple weighted t-test.

Figure 3.

R848 induces local antibody responses after mucosal or systemic immunization. Macaques were immunised intranasally (A, E, I) or intramuscularly (B, F, J) with 200 μg KLH in combination with 500 μg R848 (n = 4 per group). Immunizations were administered at weeks 0, 4 and 8. KLH specific ELISA were performed for IgG (A, B, C) and IgA (E, F, G) in nasal lavage and IgG in vaginal samples (I, J, K). Relative coefficients of excretion (RCE) compared to albumin in nasal and vaginal samples (D, H, L). Data is presented as individual animals. Pooled data for each route is presented for each sample (C, F, I), where each point represents mean of n = 4 animals.

Figure 4.

R848 induces mild local inflammation. Macaques were immunised intranasally with R848 or PBS and Nasal swabs collected at time 0, 1, 2, 3, 4, 6, 8, 24, 48, and 96 hrs. Nasal swabs examined for cellular infiltrates, neutrophils by cytospin (A), monocytes (B) and CD3 cells (C) by flow cytometry and fluids for cytokine/chemokine responses by CBA (D–I). Points represent n = 15 animals in the R848 group and 5 animals in the PBS control group +SEM, * p < 0.05 by multiple weighted t test.