Alphavirus replicon-based strategies for vaccination

Vaccination has been extremely successful for the control of many infectious diseases. However, efficient vaccines are still not available against diseases such as HIV, hepatitis C, malaria and tuberculosis. For these diseases, the traditional vaccine approaches are not feasible, and thus new vaccine technologies are needed. Some platforms that are being developed for this purpose include virus vectors and DNA vaccines. In addition, combining different vaccine modalities into heterologous prime-boost regimens induces antigen-specific immune responses that are greatly increased compared to homologous prime-boost immunization. In this thesis, we evaluated the use of alphavirus replicons as a vaccine platform and characterized antigen-specific immune responses induced in mice and rhesus macaques. Alphavirus replicons can be administered either as viral particles (VREP), or as naked DNA (DREP) or RNA (RREP). We used both model antigens (papers II and IV) and HIV antigens that are in clinical development (papers I and III). We show in paper I that antigen-specific CD8 T cell responses induced by DREP can be further increased by delivery with intradermal electroporation. These responses were superior in magnitude to those induced by a conventional DNA vaccine and required lower doses. We also showed in mice and macaques that priming with DREP rather than conventional DNA prior to a heterologous boost with a poxvirus or adenovirus vector resulted in stronger immune responses characterized by multifunctional T cells. In paper II, we characterize the kinetics and memory phenotypes of CD8 T cell responses induced by VREP and DREP. We show how altering factors such as timing and dose affects the magnitude and phenotype of the resulting immune response. In addition, we characterize the phenotypes of T cell responses induced by different heterologous boosters given after a DREP prime. For example, a poxvirus vector boost favored expansion of effector memory T cells. In paper III, we expand the heterologous prime-boost studies and explore the outcome of altering the number of DREP prime immunizations prior to a poxvirus and/or protein antigen boost. We demonstrate that a single prime with a low dose of DREP was sufficient for induction of antigen-specific T cells that were expanded by a poxvirus boost and antibody responses boosted by protein antigen. By boosting with poxvirus together with protein, both arms of adaptive immunity were induced. In paper IV, we use VREP as an adjuvant for antibody responses against a co-immunized protein antigen. We demonstrate that incorporating the innate immune stimulant flagellin into the replicon enhances its adjuvant potency, resulting in augmented antigen-specific antibody responses. In conclusion, we have characterized immune responses induced by alphavirus replicons administered as VREP or DREP and shown that DREP is an excellent prime of T cell and antibody responses prior to a heterologous boost immunization. These results strongly support further clinical testing of alphavirus replicon vaccines. LIST OF SCIENTIFIC PAPERS I. Knudsen ML, Mbewe-Mvula A, Rosario M, Johansson DX, Kakoulidou M, Bridgeman A, Reyes-Sandoval A, Nicosia A, Ljungberg K, Hanke T, Liljestrom P. 2012. Superior induction of T cell responses to conserved HIV1 regions by electroporated alphavirus replicon DNA compared to that with conventional plasmid DNA vaccine. J. Virol. 86:4082–4090. II. Knudsen ML*, Ljungberg K*, Kakoulidou M, Kostic L, Hallengard D, Garcia-Arriaza J, Merits A, Esteban M, Liljestrom P. 2014. Kinetic and phenotypic analysis of CD8 T cell responses after priming with alphavirus replicons and homologous or heterologous booster immunizations. J. Virol. 88:12438-12451. * These authors contributed equally III. Knudsen ML, Ljungberg K, Tatoud R, Weber J, Esteban M, Liljestrom P. Alphavirus replicon DNA expressing HIV antigens is an excellent prime for boosting with recombinant modified vaccinia Ankara (MVA) or with HIV gp140 protein antigen. Submitted manuscript. IV. Knudsen ML, Johansson DX, Kostic L, Nordstrom EKL, Tegerstedt K, Pasetto A, Applequist SE, Ljungberg K, Sirard J-C, Liljestrom P. 2013. The adjuvant activity of alphavirus replicons is enhanced by incorporating the microbial molecule flagellin into the replicon. PLoS One 8:e65964.