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Until recently, malaria vaccine development efforts have focused almost exclusively on a handful of well characterized Plasmodium falciparum antigens. Despite dedicated work by many researchers on different continents spanning more than half a century, a successful malaria vaccine remains elusive. Sequencing of the P. falciparum genome has revealed more than five thousand genes, providing the foundation for systematic approaches to discover candidate vaccine antigens. We are taking advantage of this wealth of information to discover new antigens that may be more effective vaccine targets. Herein, we describe different approaches to large-scale screening of the P. falciparum genome to identify targets of either antibody responses or T cell responses using human specimens collected in Controlled Human Malaria Infections (CHMI) or under conditions of natural exposure in the field. These genome, proteome and transcriptome based approaches offer enormous potential for the development of an efficacious malaria vaccine., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Modified Vaccinia virus Ankara (MVA) is an attenuated strain of vaccinia virus that is being considered as a safer alternative to replicating vaccinia vaccine strains such as Dryvax(®) and ACAM2000. Its excellent safety profile and large genome also make it an attractive vector for the delivery of heterologous genes from other pathogens. MVA was attenuated by prolonged passage through chick embryonic fibroblasts in vitro. In human and most mammalian cells, production of infectious progeny is aborted in the late stage of infection. Despite this, MVA provides high-level gene expression and is immunogenic in humans and other animals. A key issue for vaccine developers is the ability to be able to monitor an immune response to MVA in both vaccinia naïve and previously vaccinated individuals. To this end we have used antibody profiling by proteome microarray to compare profiles before and after MVA and Dryvax vaccination to identify candidate serodiagnostic antigens. Six antigens with diagnostic utility, comprising three membrane and three non-membrane proteins from the intracellular mature virion, were purified and evaluated in ELISAs. The membrane protein WR113/D8L provided the best sensitivity and specificity of the six antigens tested for monitoring both MVA and Dryvax vaccination, whereas the A-type inclusion protein homolog, WR148, provided the best discrimination. The ratio of responses to membrane protein WR132/A13L and core protein WR070/I1L also provided good discrimination between primary and secondary responses to Dryvax, whereas membrane protein WR101/H3L and virion assembly protein WR118/D13L together provided the best sensitivity for detecting antibody in previously vaccinated individuals. These data will aid the development novel MVA-based vaccines., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The present study evaluates immunogenicity and protection potency of a codon-optimized GRA1 DNA vaccine, wild type GRA1 DNA vaccine and an adjuvanted recombinant GRA1 protein vaccine candidate in BALB/c mice against lethal toxoplasmosis. Of the three GRA1 vaccines tested, the recombinant GRA1 protein vaccine results reveal significant increase in immune response and prolonged survival against acute toxoplasmosis compared to DNA vaccinations. Immune response and protection conferred by codon-optimized GRA1 DNA vaccine was slightly better than wild type GRA1 DNA vaccine.

We have examined the induction of anti-tumour immunity in a murine model using a gene vaccine approach to deliver a well defined tumour antigen. The vaccines expressed the human papilloma virus type 16 (HPV 16) E7 oncoprotein, and protection was measured against HPV 16-expressing C3R tumour cell line in vivo. In control mice injected with saline, C3R cells initially formed tumours but then regressed completely. As expected, animals injected with a peptide that represents the D(b)-presented CTL epitope from E7 (RAHYNIVTF) were completely protected from tumour growth. Contrary to expectation, however, we consistently saw enhanced tumour growth, delayed regression, or tumour outgrowth in mice vaccinated with two different E7-expressing DNA vaccines. We found no evidence for loss of D(b) or K(b) class I MHC molecules from C3R cells recovered from outgrown tumours, and fluorescent MHC/peptide tetramer staining revealed E7 gene vaccination did not delete RAHYNIVTF-specific CD8(+) T cells. However, we did observe an effect on cytokine production. Splenocytes from E7 gene vaccinated animals responded to re-stimulation in vitro with C3R cells by producing IL-4 but background levels of IFN-gamma. We also observed that cytokine production and E7 peptide-specific CTL were only detectable in vaccinated animals after C3R challenge, but not after DNA priming alone. We conclude that 'prime-boosting' is necessary to observe tumour-specific T cell responses with the gene vaccine approach, but that boosting with tumour cells causes skewing of the primed cells in a T2 direction that is incompatible with protective anti-tumour immunity.

Immunostimulating complexes (ISCOMs) efficiently deliver soluble antigen into both the cytosolic (endogenous) and endosomal (exogenous) pathways of antigen processing. Cytosolic delivery to antigen-presenting cells (APCs) may therefore be useful for the stimulation and assay of class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) in vitro. In this study, mice were immunized with ISCOMs containing fusion proteins of the E6 or E7 early proteins of human papilloma virus type 11 (HPV 11) to elicit CTL. These CTL were then restimulated in vitro using APCs pulsed with the same ISCOMs, prior to cytotoxicity assay using syngeneic target cells infected with recombinant vaccinia viruses. In this way, antigen-specific, MHC-restricted lysis by CD8+ cells was detected. However, this was dependent on the use of low density splenocytes as APCs for restimulation in vitro. Limiting dilution analyses showed a direct correlation between the CTL responder frequency and the number of times the animals were immunized in vivo. We conclude that in lieu of infectious virus, the use of ISCOMs to mediate antigen delivery to APCs in vitro can be used to quantitate CTL activity. This may have applications in monitoring vaccine efficacy, particularly to viruses such as HPV, which cannot be presently obtained as infectious virus in sufficient quantity for CTL propagation and assay.