Transgene Optimization, Immunogenicity and In Vitro Efficacy of Viral Vectored Vaccines Expressing Two Alleles of Plasmodium falciparum AMA1

Immunogenicity and In Vitro Efficacy of Viral Vectored Vaccines Expressing Two Alleles of Plasmodium falciparum AMA1. PLoS ONE 6(6): e20977. doi:10.1371/journal.pone.0020977 Transgene Optimization, Immunogenicity and In Vitro Efficacy of Viral Vectored Vaccines Expressing Two Alleles of Plasmodium falciparum AMA1 Sumi Biswas 0 Matthew D. J. Dicks 0 Carole A. Long 0 Edmond J. Remarque 0 Loredana Siani 0 Stefano 0 Colloca 0 Matthew G. Cottingham 0 Anthony A. Holder 0 Sarah C. Gilbert 0 Adrian V. S. Hill 0 Simon J. 0 Vasee Moorthy, World Health Organization, Switzerland 0 1 The Jenner Institute, University of Oxford , Oxford, Oxfordshire , United Kingdom , 2 Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases/National Institutes of Health , Rockville, Maryland , United States of America, 3 Department of Parasitology, Biomedical Primate Research Center , Rijswijk , The Netherlands, 4 Okairo` s AG, Rome , Italy , 5 Divison of Parasitology, National Institute for Medical Research , London , United Kingdom Background: Apical membrane antigen 1 (AMA1) is a leading candidate vaccine antigen against blood-stage malaria, although to date numerous clinical trials using mainly protein-in-adjuvant vaccines have shown limited success. Here we describe the pre-clinical development and optimization of recombinant human and simian adenoviral (AdHu5 and ChAd63) and orthopoxviral (MVA) vectors encoding transgene inserts for Plasmodium falciparum AMA1 (PfAMA1). Methodology/Principal Findings: AdHu5-MVA prime-boost vaccination in mice and rabbits using these vectors encoding the 3D7 allele of PfAMA1 induced cellular immune responses as well as high-titer antibodies that showed growth inhibitory activity (GIA) against the homologous but not heterologous parasite strains. In an effort to overcome the issues of PfAMA1 antigenic polymorphism and pre-existing immunity to AdHu5, a simian adenoviral (ChAd63) vector and MVA encoding two alleles of PfAMA1 were developed. This antigen, composed of the 3D7 and FVO alleles of PfAMA1 fused in tandem and with expression driven by a single promoter, was optimized for antigen secretion and transmembrane expression. These bi-allelic PfAMA1 vaccines, when administered to mice and rabbits, demonstrated comparable immunogenicity to the mono-allelic vaccines and purified serum IgG now showed GIA against the two divergent strains of P. falciparum encoded in the vaccine. CD8+ and CD4+ T cell responses against epitopes that were both common and unique to the two alleles of PfAMA1 were also measured in mice. Conclusions/Significance: Optimized transgene inserts encoding two divergent alleles of the same antigen can be successfully inserted into adeno- and pox-viral vaccine vectors. Adenovirus-MVA immunization leads to the induction of T cell responses common to both alleles, as well as functional antibody responses that are effective against both of the encoded strains of P. falciparum in vitro. These data support the further clinical development of these vaccine candidates in Phase I/IIa clinical trials. - Funding: SB was funded by MalParTraining, an FP6-funded Marie Curie Action under contract number MEST-CT-2005-020492. The GIA work was supported in part by the PATH-MVI Malaria Vaccine Initiative (MVI) and the Intramural Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases and in part by the EMVDA (European Malaria Vaccine Development Association, a European Commission FP6-funded consortium). AAH is funded by the UK Medical Research Council (U117532067). AVSH and SCG are Jenner Investigators and are funded by the Wellcome Trust. SJD is a Junior Research Fellow of Merton College, Oxford University. The funders (Okairo` s) had a role in study design. No additional external funding was received for this study. Competing Interests: SJD, SCG and AVSH are named inventors on patent applications covering malaria vectored vaccines and immunization regimes. Authors from Okairo` s are employees of and/or shareholders in Okairo` s, which is developing vectored malaria vaccines. This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials. It is estimated that in Africa alone there are 200 million cases and 0.8 million deaths due to malaria every year [1]. Vaccines remain one of the most effective public health interventions to reduce morbidity and mortality. The worldwide eradication of smallpox was due to vaccination and other infectious diseases (such as poliomyelitis and rinderpest of cattle) are similarly on the brink of eradication. In the case of malaria the development of a highly effective vaccine remains a high priority. Vaccine-induced immunity in an endemic area could help to significantly reduce overall transmission by inducing herd immunity when combined with pre-existing partially effective control interventions including insecticide-treated bednets, artemisinin-based combination therapies and indoor residual spraying [2]. Blood-stage malaria vaccines aim to mimic immunity that is naturally acquired in malaria endemic areas [3]. Individuals living in malaria endemic countries develop immunity by repeated exposure to the parasite and a significant proportion of this immunity is directed to antigens expressed on the blood-stage parasites or infected erythrocytes. This immunity is mostly antibody mediated [4], although more recently the importance of cellular immunity has begun to be recognized in mice [5,6] and humans [7,8]. Blood-stage malaria subunit vaccine development has thus mainly focused on antibody-inducing protein-in-adjuvant formulations targeting well-studied merozoite antigens, including merozoite surface proteins (MSPs) and antigens secreted from apical organelles of the parasite during erythrocyte invasion, such as apical membrane antigen 1 (AMA1). To date there have been over 30 phase I/II clinical trials with these blood-stage antigens reported [3], but the limited success of such candidates, combined with reports of vaccine allele-specific efficacy in field trials [9] and in in vitro assays of purified IgG growth inhibitory activity (GIA) [10,11], indicates that blood-stage vaccines may need to include multiple alleles of the same antigen to achieve significant efficacy against the many strains of P. falciparum in the field. PfAMA1 has been one of the leading blood-stage malaria vaccine candidate antigens for a considerable time, and there have been numerous pre-clinical and clinical AMA1 vaccine studies (reviewed in Ref [12]). Field studies have primarily addressed the importance of antibodies to PfAMA1 to clinical immunity, showing that in naturally exposed individuals the prevalence of PfAMA1-specific IgG increases with age and that this is associated with reduced risk of clinical malaria [13,14,15]. However, the PfAMA1 antigen is polymorphic, probably as a result of immune selection operating on this (...truncated)