Transgenic chloroplasts are efficient sites for high-yield production of the vaccinia virus envelope protein A27L in plant cells.

Orthopoxviruses (OPVs) have recently received increasing attention because of their potential use in bioterrorism and the occurrence of zoonotic OPV outbreaks, highlighting the need for the development of safe and cost-effective vaccines against smallpox and related viruses. In this respect, the production of subunit protein-based vaccines in transgenic plants is an attractive approach. For this purpose, the A27L immunogenic protein of vaccinia virus was expressed in tobacco using stable transformation of the nuclear or plastid genome. The vaccinia virus protein was expressed in the stroma of transplastomic plants in soluble form and accumulated to about 18% of total soluble protein (equivalent to approximately 1.7 mg/g fresh weight). This level of A27L accumulation was 500-fold higher than that in nuclear transformed plants, and did not decline during leaf development. Transplastomic plants showed a partial reduction in growth and were chlorotic, but reached maturity and set fertile seeds. Analysis by immunofluorescence microscopy indicated altered chlorophyll distribution. Chloroplast-synthesized A27L formed oligomers, suggesting correct folding and quaternary structure, and was recognized by serum from a patient recently infected by a zoonotic OPV. Taken together, these results demonstrate that chloroplasts are an attractive production vehicle for the expression of OPV subunit vaccines

Fowlpox virus recombinants expressing HPV-16 E6 and E7 oncogenes for the therapy of cervical carcinoma elicit humoral and cell-mediated responses in rabbits

Background: Around half million new cases of cervical cancer arise each year, making the development of an effective therapeutic vaccine against HPV a high priority. As the E6 and E7 oncoproteins are expressed in all HPV-16 tumour cells, vaccines expressing these proteins might clear an already established tumour and support the treatment of HPV-related precancerous lesions. Methods: Three different immunisation regimens were tested in a pre-clinical trial in rabbits to evaluate the humoral and cell-mediated responses of a putative HPV-16 vaccine. Fowlpoxvirus (FP) recombinants separately expressing the HPV-16 E6 (FPE6) and E7 (FPE7) transgenes were used for priming, followed by E7 protein boosting. Results: All of the protocols were effective in eliciting a high antibody response. This was also confirmed by interleukin-4 production, which increased after simultaneous priming with both FPE6 and FPE7 and after E7 protein boost. A cell-mediated immune response was also detected in most of the animals. Conclusion: These results establish a preliminary profile for the therapy with the combined use of avipox recombinants, which may represent safer immunogens than vaccinia-based vectors in immuno-compromised individuals, as they express the transgenes in most mammalian cells in the absence of a productive replication

Updates and achievements in virology

The 4th European Congress of Virology, hosted by the Italian Society for Virology, attracted approximately 1300 scientists from 46 countries worldwide. It also represented the first conference of the European Society for Virology, which was established in Campidoglio, Rome, Italy in 2009. The main goal of the meeting was to share research activities and results achieved in European virology units/institutes and to strengthen collaboration with colleagues from both western and developing countries. The worldwide representation of participants is a testament to the strength and attraction of European virology. The 5-day conference brought together the best of current virology; topics covered all three living domains (bacteria, archaea and eucarya), with special sessions on plant and veterinary virology as well as human virology, including two oral presentations on mimiviruses. The conference included five plenary sessions, 31 workshops, one hepatitis C virus roundtable, ten special workshops and three poster sessions, as well as 45 keynote lectures, 191 oral presentations and 845 abstracts. Furthermore, the Gesellschaft fur Virologie Loeffler-Frosch medal award was given to Peter Vogt for his long-standing career and achievements; the Gardner Lecture of the European Society for Clinical Virology was presented by Yoshihiro Kawaoka, and the Pioneer in Virology Lecture of the Italian Society for Virology was presented by Ulrich Koszinowski

Construction and characterisation of a recombinant fowlpox virus that expresses the human papilloma virus L1 protein

<p>Abstract</p> <p>Background</p> <p>Human papilloma virus (HPV)-16 is the most prevalent high-risk mucosal genotype. Virus-like-particle (VLP)-based immunogens developed recently have proven to be successful as prophylactic HPV vaccines, but are still too expensive for developing countries. Although vaccinia viruses expressing the HPV-16 L1 protein (HPV-L1) have been studied, fowlpox-based recombinants represent efficient and safer vectors for immunocompromised hosts due to their ability to elicit a complete immune response and their natural host-range restriction to avian species.</p> <p>Methods</p> <p>A new fowlpox virus recombinant encoding HPV-L1 (FP_L1) was engineered and evaluated for the correct expression of HPV-L1 <it>in vitro</it>, using RT-PCR, immunoprecipitation, Western blotting, electron microscopy, immunofluorescence, and real-time PCR assays.</p> <p>Results</p> <p>The FP_L1recombinant correctly expresses HPV-L1 in mammalian cells, which are non-permissive for the replication of this vector.</p> <p>Conclusion</p> <p>This FP_L1recombinant represents an appropriate immunogen for expression of HPV-L1 in human cells. The final aim is to develop a safe, immunogenic, and less expensive prophylactic vaccine against HPV.</p

Comparative analysis of immune responses and cytokine profiles elicited in rabbits by the combined use of recombinant fowlpox viruses, plasmids and virus-like particles in prime-boost vaccination protocols against SHIV

Three different prime-boost immunization protocols were tested in rabbits and their immune response was evaluated and compared with the final aim of identifying a vaccine strategy that might be able to protect non-human primates from infection with the pathogenic chimera simian/human immunodeficiency virus (SHIV)(89.6P). Protocols were based on priming with two fowlpox (FP) recombinant vectors and two expression plasmids, which express either the simian immunodeficiency virus (SIV)mac(239) gag/pol or the human immunodeficiency virus (HIV-1)env(89.6P) genes, followed by boosting with virus-like particles (VLP). All protocols were effective in eliciting homologous neutralizing Ab and highlighted the efficacy of VLP boosting. The FP vector was less efficient than plasmid DNA in inducing Ab against the gag core proteins. Analysis of cytokine expression 5 months after last immunization indicated that priming with pcDNA3gag/pol(SIV) and FPenv(89.6P) followed by VLP boosting generated a T helper (Th0) profile and a good Ab titer, suggesting a potential protocol to be tested in the SHIV-macaque model of HIV-1 infection