Meeting Report

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Recombinant vaccines
Expert Rev. Vaccines 9(5), 461463 (2010)

Ross Thomas Barnard

Centre for Infectious Disease Research, School of Chemistry and Molecular Biosciences, The University of Queensland, Saint Lucia, Brisbane, Queensland, 4072, Australia rossbarnard@uq.edu.au

Recombinant Vaccines: Strategies for Candidate Discovery and Vaccine Delivery BioPark Hertfordshire, Welwyn Garden City, UK, 12 March 2010
The Recombinant Vaccines: Strategies for Candidate Discovery and Vaccine Delivery conference, organized by EuroSciCon, hosted a group of UK-based and international scientists from as far afield as Malaysia and Australia. Genomic analyses of pathogens and elucidation of mechanisms of pathogenesis has advanced candidate discovery and development of vaccines. Therefore, it was timely that this conference featured, in addition to detailed expositions of target selection and clinical trials, presentations on manufacturability, scale-up and delivery of vaccines. Ten talks were presented. This meeting report describes the key topics presented and the themes that emerged from this conference.

The introduction by the Chair (Sarah Gilbert, Jenner Institute, University of Oxford, UK) set the tone for the meeting. It emphasized the need for parallel development of animal and human vaccines in order to maximize opportunities for the combination of knowledge from these fields. She stressed the importance of using appropriate target animals for challenge experiments and remarked on the recent sharp increase in interest in R&D of vaccines.
Vaccine production process
Development, manufacturing & stability

The initial focus of the meeting was on issues that are generally considered in the final stages, but, as emphasized by the speakers, should be considered early in the pathway to vaccine development. It was, therefore, appropriate that the meeting began with two presentations focussed on the development and scale-up end of the vaccine pipeline. David Simpson (Eden Biodesign, Liverpool, UK) stressed the importance of analytics and orthogonal approaches to analytics in the preparation of vaccine products for Phase I or later stage trials. He illustrated the difficulties in analytics for complex products, pointing out that the complexity in vaccine products needs to be retained in the scaled-up production processes and that the retention of complexity needs to be demonstrable, entailing the use of a suite of analytic techniques. He made the salient point that if a Phase I clinical
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trial is the objective, a Phase I process should be developed, not a Phase III. Case studies in the production of virus-like particles and adenovirus were presented to illustrate the production and analytical approaches that have been used to bring products to the Phase I clinical trial stage. R&D work to ensure efficacy and manufacturability will be wasted if product stability cannot be assured under temperature conditions prevailing in emerging countries. This is particularly important at a time of vaccine stockpiling. Stephen Ward (Stabilitech, Imperial Incubator, Imperial College London, UK), presented data on performance of a panel of low-cost, nontoxic, water-soluble excipients that were effective in long-term stabilization of live viral vaccines and biopharmaceuticals. He reported that the individual components had been safely used previously in clinical settings, and could be easily integrated into existing cGMP manufacturing processes. Ward also described an efficient multivariate design of experiments approach to rapidly determine optimal mixes of excipients.
New foot-and-mouth disease vaccines

The challenge of vaccine stability was again raised in the presentation by David Paton (Institute for Animal Health, Pirbright, UK) on the prospects for improved vaccines against footand-mouth disease. A total of 23 billion doses of the current vaccines are consumed annually and there is a requirement for emergency stockpiles;
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Barnard

however, the antigen is unstable and must be stored under liquid nitrogen. Paton discussed novel molecular strategies for the production of stable virus-like particles containing multiple antigenic epitopes, but lacking nucleic acids. Such empty capsid vaccines have the potential to allow clean distinction between vaccinated animals and infected animals, by utilizing diagnostic tests for nonstructural (NS) proteins, since no NS proteins are present in an empty capsid vaccine. These vaccines offer the potential of better shelf life, safer production and better long-term immunity. The combination of DNA vaccines with a protein-antigen boost was also described.
Investigating T- & B-cell contributions to immunity against nontyphoid Salmonella

Nontyphoid Salmonella (NTS) causes lethal bacteremia in children and HIV patients in Africa. Currently, there is no vaccine against NTS and typhoid vaccines do not cross-protect. Adriana Flores-Langarica (MRC Centre for Immune Regulation, School of Immunity and Infection, University of Birmingham, UK) presented elegant basic research on the cellular basis of immune responses, while identifying promising leads for new NTS vaccines. Porins are conserved membrane proteins found in Gramnegative bacteria. Immunization of mice with porin induces T-cellindependent responses with similar kinetics to live-attenuated Salmonella typhimurium. Highly purified porins confer protection equivalent to a heat-killed vaccine. An important observation was that HIV-positive patients who fail to kill the bacteria have higher anti-lipopolysaccharide IgG titers. A striking experimental result was shown in which addition of anti-lipopolysaccharide antibodies to HIV-negative sera inhibited the killing of bacteria. Flagellin induces potent antibody responses in mice but this does not contribute to clearance of bacteria. Despite the induction of Th2 features, flagellin-promoted bacterial clearance is T-bet mediated. The presentation brought home the point that resolution of Salmonella infection is dependent upon correct timing of the different elements of the immune response.
Regulatory challenges, new ways to deliver vaccines & mucosal immunity

Genetically modified vaccines have great potential to induce cellmediated immunity and antibody responses targeting mucosal surfaces. David Lewis (St Georges Hospital, University of London, UK) discussed the complex challenges of regulatory compliance-facing investigators planning first-time-in-humans trials of genetically modified vaccines. Case studies were presented on completed Phase I and II trials of the live oral vaccine against Shigella dysenteriae type 1, developed using a rational attenuation strategy based on detailed knowledge of molecular pathogenesis. Research towards a drinkable vaccine against Salmonella typhi was described. Experiments in mice have been carried out using a fascinating technique called sequence tag mutagenesis, which entails random gene knockouts by insertion of molecular barcodes. The absence of particular barcodes from the shed population of Salmonella enabled discovery of deletions associated with attenuation. Phase I trials with attenuated S. typhimurium and
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S. typhi aroC- ssaV- have allowed progress to translational development. Earlier studies on the use of orally delivered, live-attenuated (SPI-2 deleted) genetically modified vaccines to deliver a heterogeneous antigen (Escherichia coli heat-labile toxin B) have shown that additional work is needed to optimize immune response to the antigen presented in the Salmonella background. Continuing the theme of mucosal delivery, Valerie Ferro (University of Strathclyde, Glasgow, Scotland) reported the development of a vaccine delivery system consisting of nonionic surfactant vesicles and bile salts (bilosomes), which protect the antigen against gastrointestinal degradation. The delivery system has been tested with an orally administered influenza A vaccine in rodents, ferrets and primates, and is now ready for Phase I clinical trials. The large bilosomes resulted in a Th1-biased response and greater protection, as measured by symptom score in the ferret model. The future application of bilosome delivery to fertility control by immunization with sperm antigens was discussed. The impact of modes of delivery on stimulation of mucosal immunity was the key theme of the presentation by Robin Shattock (St Georges Hospital Medical School, University of London, UK), on optimizing vaginal immunity to HIV-1. The work showed the critical importance of the route of vaccine administration and boost on the strength of vaginal immune response. He reported on complex clinical and paraclinical trials in nonhuman primates, designed to assess responses to the gp140 CN54 trimer (his groups previous work showed this antigen to be stable on the vaginal mucosal surface). Initial human studies using repeat intravaginal administration showed a lack of reproducible mucosal responses and no detectable systemic response. Testing in nonhuman primates showed both systemic and mucosal responses to single intramuscular prime, boosted by a single round of intravaginal immunization. Studies in mice suggested that intra nasal administration of vaccine with a novel adjuvant, followed by intranasal boost may be the most effective means of producing IgG and vaginal IgA responses. New clinical and paraclinical trials in nonhuman primates are underway to test this combination and various modes of administration. Michael Skinner (Vaccine Vector Group, Imperial College London) presented an overview of the history of safe use of recombinant fowlpox virus and modified vaccinia Ankara (MVA) virus vaccine vectors in chickens and mammals, including humans. Trovac-Avian influenza live recombinant vaccine was widely used in chickens in Mexico in 19951996 to combat high pathogenicity avian influenza. He reviewed work on prime-boost studies in mice using fowlpox virus (FP9)Plasmodium berghei circumsporozoite protein and MVA-CSP recombinants. Coadministration of IL-18 enhanced the immune response and reduced the bursal load of infectious bursal disease virus in chickens vaccinated with recombinant fowlpox virus expressing infectious bursal disease virus antigen. Large-scale genetic screening studies have identified genes encoding the resistance of FP9 virus to avian interferon, and genes that block the synthesis of chicken IFN-b in vitro. Candidates have been identified and additional studies have been planned to elucidate the molecular mechanisms of
Expert Rev. Vaccines 9(5), (2010)

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Meeting Report

these processes that are of fundamental importance to our understanding of viral pathogenesis and the interactions of viruses with the host immune system.
New vaccines against old enemies

Despite efforts taken to control Vibrio cholerae infection, the global burden of cholera remains high, mostly affecting young children. Murugaiah Chandrika (Universiti Sains Malaysia, Penang, Malaysia) presented work on the construction and evaluation of a new, genetically modified, live-attenuated vaccine (VCUSM21P) with a view to facilitate rapid mass vaccination. Construction of this multiple mutant of V. cholerae O139, and evaluation of its cytotoxicity and reactogenicity were described. Oral immunization of rabbits produced protective and vibriocidal IgA and IgG responses. Elegant electron microscopy and histochemical studies of immunized and nonimmunized rabbits were presented to demonstrate the lack of intestinal colonization in vaccinated animals. VCUSM21P was found to be suitably nonreactogenic and immunogenic in vivo, protecting rabbits from lethal challenge. This new genetically modified vaccine has the potential to reduce cholera disease burdens and childhood mortality. The WHO has stated that development of a pandemic influenza vaccine in the fastest possible time is a global priority [1] . Gilbert reviewed the current formulations of influenza A vaccines and the challenge presented by the need to reformulate the vaccine each season. Work by McMicheal et al. was cited, demonstrating that cytotoxic T lymphocytes acquired by natural infection in humans can protect against subsequent exposure, even providing cross-subtype protection [2] . Other work (Lee et al.) reviewed by Gilbert, has shown that the highly conserved nucleoprotein (NP) and matrix (M)1 proteins are recognized strongly in the immune response to influenza A [3] . Most adults have been exposed to influenza
References
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A and have some T-cell memory against NP and M1, so it was postulated that single, low-dose immunization with recombinant MVA expressing a NP:M1 fusion should boost these to potentially protective levels. Safety and immunogenicity studies have been carried out and challenge studies in humans are in progress. The vaccine is produced in suspension cells. The final formulation will be lyophilized and stabilized for room temperature storage. It will not require reformulation each influenza season and manufacturing could be carried out year round.
Conclusion

The conference presented the latest work addressing the key challenges in recombinant vaccine research: stability, delivery, and elicitation of strong systemic and mucosal immune responses. Gilbert summed up the meeting by noting that the presentations had highlighted the importance of understanding the molecular basis of disease and immune responses, the importance of careful trial design and the use of appropriate models for testing. The feasibility of translating research to the clinic needs to be considered at the earliest stages of research.
Acknowledgements

This meeting was organized by Euroscicon (www.euroscicon.com).

Financial & competing interests disclosure

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
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Collin N, de Radigus X; the WHO H1N1 Vaccine Task Force. Vaccine production capacity for seasonal and pandemic (H1N1) 2009 influenza. Vaccine 27, 51845186 (2009).

McMichael AJ, Gotch FM, Noble GR, Beare PA. Cytotoxic T-cell immunity to influenza. N. Engl. J. Med. 309, 1317 (1983).

Lee LY-H, Ha DLH, Simmons C et al. Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals. J. Clin. Invest. 118(10), 34783490 (2008).

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