Innate Immune Responses in Vivo after Antigen Administration – Implications for Vaccine Development–

Inducing high magnitude of antibodies with epitope breadth over prolonged periods of time is likely a prerequisite to prevent several of the world’s most serious infectious diseases such as HIV-1, malaria and tuberculosis for which there are no vaccines yet. A much better understanding of the innate immune mechanisms that are critical for inducing strong responses to vaccination is therefore essential. The overall aim of this thesis was to characterize early innate immune responses in vivo after administration of antigens. This includes studies of the recruitment of immune cell subsets to the site of antigen injection (e.g. skin or muscle), local cell activation and presence of inflammatory mediators, antigen uptake and transport and finally initiation of adaptive immunity in lymph nodes (LNs). To be able to approach this in humans in vivo, we first utilized skin punch biopsies collected from sites injected with purified protein derivate (PPD), which is a mixture of mycobacterial antigens used in the tuberculin skin test (TST). By performing tissue staining of cryosections we show in paper I that several subsets of dendritic cells (DCs), including the plasmacytoid DCs (PDCs) normally not residing in skin, infiltrated the dermis in the positive TST indurations, which was in contrast to donormatched saline-injected skin. The positive TST indurations were associated with cell death and high expression of the antimicrobial peptide LL37, which together can provide means for PDC activation and IFNα production. In line with this, IFN-inducible MxA was highly expressed in the positive TST sites. We expanded the studies in paper II and DC accumulation was also found in skin biopsies taken after skin tests using antigens from either mumps virus or Candida albicans. Further, TST indurations of HIV-1 individuals also showed DC infiltration but to a lower degree, which likely reflect on the reduced integrity of their immune system. To this end, the level of DCs in the positive TST reactions correlated with the level of infiltrating T cells. The skin antigen tests represent recall of immunological memory responses locally. To enable studies of local innate immune activation after vaccine administration and priming of naïve responses, we developed a nonhuman primate (NHP) model in the second part of the thesis. After establishing and validating protocols for sample collection and tissue processing in paper III, we examined in paper IV how the distinct vaccine adjuvants; alum (benchmark), MF59 (emulsion) or alum with TLR7 agonist, influence the innate responses leading to adaptive immunity. HIV-1 envelope glycoprotein (Env) was administered as the vaccine antigen together with the adjuvants. We found a rapid infiltration of neutrophils, monocytes and DCs to the vaccine-injected muscle with all adjuvants. Env cells were readily detected in the muscle and draining LNs. In line with the finding of alum-TLR7 and MF59 being superior over alum in terms of inducing both antibodyand T cell responses, alum consistently showed lower innate activation. While alum-TLR7 consistently induced robust DC maturation and type IFN I responses, MF59 induced neutrophil homing to LNs. Comparison of antigen presentation capacity of Env cells in the draining lymph nodes showed that myeloid DCs exceled at stimulating Env-specific CD4 T cell responses. However, neutrophils were also capable of antigen presentation. Despite inducing different innate activation, both MF59 and alum-TLR7 enhanced the priming of Env-specific T cells in vaccine-draining LNs as well as increased the differentiation of T follicular helper cells and germinal center formations compared to alum. In summary, our findings demonstrate the initial immune events at the sites of antigen delivery, including vaccination in vivo. These early immunological responses shape the quantity and quality of adaptive immunity. Understanding the mechanisms by which distinct adjuvants influence vaccine response will help in the selection of the best-suited adjuvant to improve vaccine efficacy to a given pathogen. LIST OF SCIENTIFIC PAPERS I. Emily Bond*, Frank Liang*, Kerrie J Sandgren, Anna Smed-Sörensen, Peter Bergman, Susanna Brighenti, William C Adams, Senait A Betemariam, Molebogeng X Rangaka, Christopher Lange, Robert J Wilkinson, Jan Andersson and Karin Loré. Plasmacytoid denritic cells infiltrate the skin of positive tuberculin skin test indurations. *shared first authorship. Journal of Investigative Dermatology 2012;132(1):114-23 II. Frank Liang, Emily Bond, Kerrie J Sandgren, Anna Smed-Sörensen, Molebogeng X Rangaka, Christoph Lange, Richard A Koup, Grace A McComsey, Michael M Lederman, Robert J Wilkinson, Jan Andersson and Karin Loré. Dendritic cell recruitment in response to skin antigen tests in HIV-1-infected individuals correlates with the level of T-cell infiltration. AIDS 2013, 27:1071-1080 III. Frank Liang*, Aurélie Ploquin*, José DelaO Hernández, Hugues Fausther-Bovendo, Gustaf Lindgren, Daphne Stanley, Aiala Salvador Maritinez, Jason M Brenchley, Richard A Koup, Karin Loré** and Nancy J Sullivan**. Dissociation of skeletal muscle for flow cytometric characterization of immune cells in macaques. *shared first authorship, **shared last authorship Journal of Immunological Methods, 2015, S0022-1759(15)30013-2. DOI:10.1016/j.jim.2015.06.011. IV. Frank Liang, Kerrie J Sandgren, Gustaf Lindgren, Elizabeth A Thompson, Joseph R Francica, Anja Seubert, Ennio De Gregorio, Susan Barnett, Derek O’Hagan, Srinivas Rao, Nancy J Sullivan, Richard A Koup, Robert A Seder and Karin Loré. The adjuvants MF59 and Alum combined with TLR7 agonist differentially stimulate neutrophil and dendritic cell functions leading to enhanced vaccine responses. Manuscript Publications not included in this thesis: Elizabeth A Thompson, Frank Liang, Gustaf Lindgren, Kerrie J Sandgren, Kylie M Quinn, Patricia A Darrah, Richard A Koup, Robert A Seder, Ross M Kedl, Karin Loré. Human anti-CD40 antibody and poly IC:LC adjuvant combination induces potent T cell responses in the lung of non-human primates. Journal of Immunology, 2015, 1;195(3):1015-24. Chuanfeng Wu, Brian Li, Rong Lu, Samson J Koelle, Yanqin Yang, Alexander Jares, Alan E Krouse, Mark Metzger, Frank Liang, Karin Loré, Colin O Wu, Robert E Donahue, Irvin SY Chen, Irving Weissman, Cynthia E Dunbar. Clonal tracking of rhesus macaque hematopoiesis highlights a distinct lineage origin for natural killer cells. Cell Stem Cell 2014, 3;14(4):486-99 Matthew J Johnson, Niklas K Björkström, Costas Petrovas, Frank Liang, Jason G Gall, Karin Loré and Richard A Koup. Type I interferondependent activation of NK cells by rAd28 or rAd35, but not rAd5, leads to loss of vector-insert expression. Vaccine 2014, 3;32(6):717-24 Kerrie J Sandgren, Frank Liang, Anna Smed-Sörensen and Karin Loré. Dendritic cell interactions with HIV-1 envelope glycoprotein Implications for preventing transmission. Book chapter in the Encyclopedia of AIDS, Springer New York 2014 Sep 20: 1-14. DOI 10.1007/978-1-4614-9610-6_133-1. ISBN 978-1-46149610-6. Kerrie J Sandgren, Anna Smed-Sörensen, Mattias N Forsell, Maritna Soldemo, Willian C Adams, Frank Liang, Leif Perbeck, Richard A Koup, Richard T Wyatt, Gunilla B Karlsson Hedestam, Karin Loré. Human plasmacytoid dendritic cells efficiently capture the HIV-1 envelope glycoprotein via CD4 for antigen presentation. Journal of Immunology 2013, 191(1):60-9.