Adjuvants are critical for the success of vaccines. Agonists of microbial pattern recognition receptors (PRRs) are promising new adjuvant candidates. A mechanism through which adjuvants enhance immune responses is to stimulate innate immunity. We studied the innate immune response in humans to synthetic double-stranded RNA (polyinosinic:polycytidylic acid [poly IC] stabilized with poly-L-lysine [poly ICLC]), an agonist for toll-like receptor (TLR) 3, and the cytosolic RNA helicase MDA-5. Transcriptional analysis of blood samples from eight volunteers, after subcutaneous administration of poly ICLC, showed up-regulation of genes involved in multiple innate immune pathways in all subjects, including interferon (IFN) and inflammasome signaling. Blocking type I IFN receptor ex vivo significantly dampened the response to poly IC. Comparative transcriptional analysis showed that several innate immune pathways were similarly induced in volunteers immunized with the highly efficacious yellow fever vaccine. Therefore, a chemically defined PRR agonist like poly ICLC can be a reliable and authentic microbial mimic for inducing innate immune responses in humans.
Protein vaccines, if rendered immunogenic, would facilitate vaccine development against HIV and other pathogens. We compared in nonhuman primates (NHPs) immune responses to HIV Gag p24 within 3G9 antibody to DEC205 ("DEC-HIV Gag p24"), an uptake receptor on dendritic cells, to nontargeted protein, with or without poly ICLC, a synthetic double stranded RNA, as adjuvant. Priming s.c. with 60 ?g of both HIV Gag p24 vaccines elicited potent CD4(+) T cells secreting IL-2, IFN-?, and TNF-?, which also proliferated. The responses increased with each of three immunizations and recognized multiple Gag peptides. DEC-HIV Gag p24 showed better cross-priming for CD8(+) T cells, whereas the avidity of anti-Gag antibodies was ?10-fold higher with nontargeted Gag 24 protein. For both protein vaccines, poly ICLC was essential for T- and B-cell immunity. To determine whether adaptive responses could be further enhanced, animals were boosted with New York vaccinia virus (NYVAC)-HIV Gag/Pol/Nef. Gag-specific CD4(+) and CD8(+) T-cell responses increased markedly after priming with both protein vaccines and poly ICLC. These data reveal qualitative differences in antibody and T-cell responses to DEC-HIV Gag p24 and Gag p24 protein and show that prime boost with protein and adjuvant followed by NYVAC elicits potent cellular immunity.
Improved protein-based vaccines should facilitate the goal of effective vaccines against HIV and other pathogens. With respect to T cells, the efficiency of immunization, or "immunogenicity," is improved by targeting vaccine proteins to maturing dendritic cells (DCs) within mAbs to DC receptors. Here, we compared the capacity of Langerin/CD207, DEC205/CD205, and Clec9A receptors, each expressed on the CD8(+) DC subset in mice, to bring about immunization of microbial-specific T cells from the polyclonal repertoire, using HIV gag-p24 protein as an antigen. ?-Langerin mAb targeted splenic CD8(+) DCs selectively in vivo, whereas ?-DEC205 and ?-Clec9A mAbs targeted additional cell types. When the mAb heavy chains were engineered to express gag-p24, the ?-Langerin, ?-DEC205, and ?-Clec9A fusion mAbs given along with a maturation stimulus induced comparable levels of gag-specific T helper 1 (Th1) and CD8(+) T cells in BALB/c × C57BL/6 F1 mice. These immune T cells were more numerous than targeting the CD8(-) DC subset with ?-DCIR2-gag-p24. In an in vivo assay in which gag-primed T cells were used to report the early stages of T-cell responses, ?-Langerin, ?-DEC205, and ?-Clec9A also mediated cross-presentation to primed CD8(+) T cells if, in parallel to antigen uptake, the DCs were stimulated with ?-CD40. ?-Langerin, ?-DEC205, and ?-Clec9A targeting greatly enhanced T-cell immunization relative to nonbinding control mAb or nontargeted HIV gag-p24 protein. Therefore, when the appropriate subset of DCs is targeted with a vaccine protein, several different receptors expressed by that subset are able to initiate combined Th1 and CD8(+) immunity.
Protein vaccines for T-cell immunity are not being prioritized because of poor immunogenicity. To overcome this hurdle, proteins are being targeted to maturing dendritic cells (DCs) within monoclonal antibodies (mAbs) to DC receptors. To extend the concept to humans, we immunized human immunoglobulin-expressing mice with human DEC205 (hDEC205) extracellular domain. 3D6 and 3G9 mAbs were selected for high-affinity binding to hDEC205. In addition, CD11c promoter hDEC205 transgenic mice were generated, and 3G9 was selectively targeted to DCs in these animals. When mAb heavy chain was engineered to express HIV Gag p24, the fusion mAb induced interferon-?- and interleukin-2-producing CD4(+) T cells in hDEC205 transgenic mice, if polynocinic polycytidylic acid was coadministered as an adjuvant. The T-cell response was broad, recognizing at least 3 Gag peptides, and high titers of anti-human immunoglobulin G antibody were made. Anti-hDEC205 also improved the cross-presentation of Gag to primed CD8(+) T cells from HIV-infected individuals. In all tests, 3D6 and 3G9 targeting greatly enhanced immunization relative to nonbinding control mAb. These results provide preclinical evidence that in vivo hDEC205 targeting increases the efficiency with which proteins elicit specific immunity, setting the stage for proof-of-concept studies of these new protein vaccines in human subjects.
To improve the efficacy of T cell-based vaccination, we pursued the principle that CD4(+) T cells provide help for functional CD8(+) T cell immunity. To do so, we administered HIV gag to mice successively as protein and DNA vaccines. To achieve strong CD4(+) T cell immunity, the protein vaccine was targeted selectively to DEC-205, a receptor for antigen presentation on dendritic cells. This targeting helped CD8(+) T cell immunity develop to a subsequent DNA vaccine and improved protection to intranasal challenge with recombinant vaccinia gag virus, including more rapid accumulation of CD8(+) T cells in the lung. The helper effect of dendritic cell-targeted protein vaccine was mimicked by immunization with specific MHC II binding HIV gag peptides but not peptides from a disparate Yersinia pestis microbe. CD4(+) helper cells upon adoptive transfer allowed wild-type, but not CD40(-/-), recipient mice to respond better to the DNA vaccine. The transfer also enabled recipients to more rapidly accumulate gag-specific CD8(+) T cells in the lung following challenge with vaccinia gag virus. Thus, complementary prime boost vaccination, in which prime and boost favor distinct types of T cell immunity, improves plasmid DNA immunization, including mobilization of CD8(+) T cells to sites of infection.
Relative to several other toll-like receptor (TLR) agonists, we found polyinosinic:polycytidylic acid (poly IC) to be the most effective adjuvant for Th1 CD4(+) T cell responses to a dendritic cell (DC)-targeted HIV gag protein vaccine in mice. To identify mechanisms for adjuvant action in the intact animal and the polyclonal T cell repertoire, we found poly IC to be the most effective inducer of type I interferon (IFN), which was produced by DEC-205(+) DCs, monocytes, and stromal cells. Antibody blocking or deletion of type I IFN receptor showed that IFN was essential for DC maturation and development of CD4(+) immunity. The IFN-AR receptor was directly required for DCs to respond to poly IC. STAT 1 was also essential, in keeping with the type I IFN requirement, but not type II IFN or IL-12 p40. Induction of type I IFN was mda5 dependent, but DCs additionally used TLR3. In bone marrow chimeras, radioresistant and, likely, nonhematopoietic cells were the main source of IFN, but mda5 was required in both marrow-derived and radioresistant host cells for adaptive responses. Therefore, the adjuvant action of poly IC requires a widespread innate type I IFN response that directly links antigen presentation by DCs to adaptive immunity.
Toll-like receptor (TLR) ligands are being considered as adjuvants for the induction of antigen-specific immune responses, as in the design of vaccines. Polyriboinosinic-polyribocytoidylic acid (poly I:C), a synthetic double-stranded RNA (dsRNA), is recognized by TLR3 and other intracellular receptors. Poly ICLC is a poly I:C analogue, which has been stabilized against the serum nucleases that are present in the plasma of primates. Poly I:C(12)U, another analogue, is less toxic but also less stable in vivo than poly I:C, and TLR3 is essential for its recognition. To study the effects of these compounds on the induction of protein-specific immune responses in an animal model relevant to humans, rhesus macaques were immunized subcutaneously (s.c.) with keyhole limpet hemocyanin (KLH) or human papillomavirus (HPV)16 capsomeres with or without dsRNA or a control adjuvant, the TLR9 ligand CpG-C. All dsRNA compounds served as adjuvants for KLH-specific cellular immune responses, with the highest proliferative responses being observed with 2 mg/animal poly ICLC (p = 0.002) or 6 mg/animal poly I:C(12)U (p = 0.001) when compared with immunization with KLH alone. Notably, poly ICLC -- but not CpG-C given at the same dose -- also helped to induce HPV16-specific Th1 immune responses while both adjuvants supported the induction of strong anti-HPV16 L1 antibody responses as determined by ELISA and neutralization assay. In contrast, control animals injected with HPV16 capsomeres alone did not develop substantial HPV16-specific immune responses. Injection of dsRNA led to increased numbers of cells producing the T cell-activating chemokines CXCL9 and CXCL10 as detected by in situ hybridization in draining lymph nodes 18 hours after injections, and to increased serum levels of CXCL10 (p = 0.01). This was paralleled by the reduced production of the homeostatic T cell-attracting chemokine CCL21. Thus, synthetic dsRNAs induce an innate chemokine response and act as adjuvants for virus-specific Th1 and humoral immune responses in nonhuman primates.
Presumptive dendritic cells (DCs) bearing the CD11c integrin and other markers have previously been identified in normal mouse and human aorta. We used CD11c promoter-enhanced yellow fluorescent protein (EYFP) transgenic mice to visualize aortic DCs and study their antigen-presenting capacity. Stellate EYFP(+) cells were readily identified in the aorta and could be double labeled with antibodies to CD11c and antigen-presenting major histocompatability complex (MHC) II products. The DCs proved to be particularly abundant in the cardiac valves and aortic sinus. In all aortic locations, the CD11c(+) cells localized to the subintimal space with occasional processes probing the vascular lumen. Aortic DCs expressed little CD40 but expressed low levels of CD1d, CD80, and CD86. In studies of antigen presentation, DCs selected on the basis of EYFP expression or binding of anti-CD11c antibody were as effective as DCs similarly selected from the spleen. In particular, the aortic DCs could cross-present two different protein antigens on MHC class I to CD8(+) TCR transgenic T cells. In addition, after intravenous injection, aortic DCs could capture anti-CD11c antibody and cross-present ovalbumin to T cells. These results indicate that bona fide DCs are a constituent of the normal aorta and cardiac valves.
Mass Spectrometry (MS) is becoming a preferred method to identify class I and class II peptides presented on major histocompability complexes (MHC) on antigen presenting cells (APC). We describe a combined computational and MS approach to identify exogenous MHC II peptides presented on mouse spleen dendritic cells (DCs). This approach enables rapid, effective screening of a large number of possible peptides by a computer-assisted strategy that utilizes the extraordinary human ability for pattern recognition. To test the efficacy of the approach, a mixture of epitope peptide mimics (mimetopes) from HIV gag p24 sequence were added exogenously to Fms-like tyrosine kinase 3 ligand (Flt3L)-mobilized splenic DCs. We identified the exogenously added peptide, VDRFYKTLRAEQASQ, and a second peptide, DRFYKLTRAEQASQ, derived from the original exogenously added 15-mer peptide. Furthermore, we demonstrated that our strategy works efficiently with HIV gag p24 protein when delivered, as vaccine protein, to Flt3L expanded mouse splenic DCs in vitro through the DEC-205 receptor. We found that the same MHC II-bound HIV gag p24 peptides, VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ, were naturally processed from anti-DEC-205 HIV gag p24 protein and presented on DCs. The two identified VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ MHC II-bound HIV gag p24 peptides elicited CD4(+) T-cell mediated responses in vitro. Their presentation by DCs to antigen-specific T cells was inhibited by chloroquine (CQ), indicating that optimal presentation of these exogenously added peptides required uptake and vesicular trafficking in mature DCs. These results support the application of our strategy to identify and characterize peptide epitopes derived from vaccine proteins processed by DCs and thus has the potential to greatly accelerate DC-based vaccine development.
Targeting antigens encoded by DNA vaccines to dendritic cells (DCs) in the presence of adjuvants enhances their immunogenicity and efficacy in mice.
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