Systemic immunization with CCL27/CTACK modulates immune responses at mucosal sites in mice and macaques

Abstract

Plasmid DNA is a promising vaccine platform that has been shown to be safe and able to be administered repeatedly without vector interference. Enhancing the potency of DNA vaccination through co-delivery of molecular adjuvants is one strategy currently under investigation. Here we describe the use of the novel chemokine adjuvant CCL27/CTACK to enhance immune responses to an HIV-1 or SIV antigen in mice and rhesus macaques. CCL27 has been shown to play a role in inflammatory responses through chemotaxis of CCR10+ cells, and we hypothesized that CCL27 may modulate adaptive immune responses. Immunizations in mice with HIV-1gag/CCL27 enhanced immune responses both at peripheral and, surprisingly, at mucosal sites. To confirm these findings in a large-animal model, we created optimized CCL27 and SIV antigenic plasmid constructs for rhesus macaques. 10 macaques (n=5/group) were immunized intramuscularly with 1mg/construct of antigenic plasmids+/-CCL27 with electroporation. We observed significant IFN-gamma secretion and CD8+ T-cell proliferation in peripheral blood. Interestingly, CCL27 co-immunized macaques exhibited a trend toward greater effector CD4+ T cells in the bronchiolar lavage (BAL). CCL27 co-delivery also elicited greater antigen-specific IgA at unique sites including BAL and fecal samples but not in the periphery. Future studies incorporating CCL27 as an adjuvant in vaccine or therapy models where eliciting immune responses in the lung are warranted.

Figure 1. Co-vaccination with murine pCCL27 enhances antigen specific IFN-γ and IgA antibodies in the periphery and mucosa

Mice were immunized intramuscularly 3 times, two weeks apart. Samples were harvested one week following the final injection from pVAX control, pHIV-1gag, or pHIV-1gag/pCCL27 immunized mice. A) Splenocytes were cultured overnight with R10 media (negative control), or HIV-1gag peptide pools 1 through 4. The bar represents the total number of HIV-1 gag specific IFN-γ spot forming cells (SFC) per million splenocytes after background subtraction. B) Lymphocytes from the mesenteric lymph nodes were harvested and stimulated with either R10 or HIV-1gag peptides. Cells were stained as described in methods and 50,000 CD3+ live lymphocytes (as determined by the LIVE/DEAD stain) were collected. Total IFN-γ secreting CD4+ (grey bar) or CD8+ (black bar). Responses from the negative control wells were subtracted from the antigenic stimulations prior to graphing. C,D) The average fold increase of anti-HIVgag-specific IgA elicited by the chemokine CCL27 adjuvant over pHIV-1gag antigenic plasmid alone is shown in panel C (sera IgA) and panel D (fecal IgA). Data from 6 independent experiments was averaged and fold increases were determined. Levels of HIV-1 specific IgA was determined by an ELISA assay against HIV-1gag p24 protein. Quantitation of fecal and serum IgA (μg/ml) was determined using a recombinant IgA standard of known concentration as the standard curve, and fold increase values were determined. E,F) CCL27 enhances IgA by B lymphocytes in the spleen (E) and gut-associated lymphoid tissue (GALT) (F). Whole splenocytes (E) or Peyer's patch (F) cells were placed in an HIV-1gag p24 protein coated, 96 well ELIspot blocked plate and incubated for 5hrs. Secreted anti-HIV-1gag IgA by effector or memory B cells is captured, counted, and graphed as HIV-1-specific IgA Antibody Secreting Cells (ASC) per million splenocytes or Peyer's patch cells. Statistical differences are noted with ** (p< 0.01) as determined by student's t-test.

Figure 2. The rhesus CCL27 plasmid encodes functional chemokine

A) Rhesus CCL27 was RNA and codon optimized for expression in rhesus macaques and inserted into a pVAX1 expression vector. B) Plasmid CCL27 expresses the appropriate size chemokine (12.4 kDa) as detected by radioactive in vitro translation. C) CCL27 protein is detectable by sandwich ELISA in the supernatant of RD cells transfected with pCCL27, but not with the pVAX control (t=48hrs).

Figure 3. Immunization with SIV antigenic constructs elicits potent IFN-γ secretion and highly proliferative CD8+T cells

A) IFN-γ ELISpot was performed on lymphocytes isolated from peripheral blood of rhesus macaques 2 weeks post each immunization. Results are graphed as IFN-γ secreting cells/10⁶ PBMCs in response to each antigen with values from media wells subtracted. Data are shown as averages for each group of macaques following each immunization. B,C) PBMCs from immunized macaques were stained with Carboxyfluorescein succinimidyl ester (CFDA-SE) and cultured for 5 days in the presence of media (negative control), SIV Gag, Env, or Pol peptides, or ConA (positive control). Cells were then stained for surface markers (CD3, CD4, CD8) and run on flow cytometry. B) An example of proliferation in an immunized macaque. C) The average proliferation of CD8+ T cells in response to each SIV antigen from groups of macaques 4 weeks after each vaccination.

Figure 4. Co-delivery of CCL27 does not enhance the magnitude of cytokine secretion in the periphery

PBMCs from immunized macaques were examined for cytokine secretion in response to stimulation with SIV peptide antigens (Gag, Pol or Env) in the presence of Golgi transport inhibitors and CD107a for 5 hours. Intracellular cytokine staining (ICS) was carried out from PBMCs following the final immunization. Data is graphed after values from media wells were subtracted. A) The total magnitude of cytokine secretion (and degranulation) was determined by adding the total number of cytokine secreting cells for each antigen for either CD4+(CD8-) or CD8+ (CD4-) T cells. The contribution of each function to the overall CD4+ and CD8+ T cell responses are shown in (B).

Figure 5. Co-delivery of CCL27 augments cytokine secretion in the lung

Levels of CD107a and IFN-γ expression by CD4+ T cells isolated from the periphery (PBMCs) or the mucosa (BAL) of immunized macaques were measured by ICS. Cells were stimulated with SIV Env for 5 hours in the presence of Golgi transport inhibitors. The percent of CD4% T cells responding minus background values in media only wells are shown.

Figure 6. DNA immunization elicits potent memory responses

Memory responses are robust in the periphery. PBMCs were isolated from immunized macaques 23 weeks following the 4^th immunization and assayed for IFN-γ secretion by ELISpot (A, B) and CD8+T cell proliferation (C). The numbers of IFN-γ secreting cells are graphed for individual vaccinated macaques (A) or as averages for each group are shown in (B). The average CD8+T cell proliferation as determined by CFSE proliferation assays are shown in (C).

Figure 7. CCL27 augments antigen-specific IgA at mucosal, but not systemic sites

Antigen specific IgG or IgA antibody levels in various samples from immunized macaques were determined by endpoint titer ELISAs and B cell ELISpot. A) Sera samples were added to plates coated with SIVmac239 Env gp130 recombinant protein or BSA and the endpoint titers of IgG were determined by the O.D value in antigen specific wells that were two-fold over BSA values. Endpoint titers of antigen specific IgA, were also measured in the sera (B), bronchial lavage (C), and fecal pellets (D) by ELISAs. Mucosal samples were taken 10 weeks post the 4^th immunization. Statistical differences are noted with a * (p< 0.05) and were determined by student's t-test.