Report from the field

Abstract

When the next pandemic emerges, will we be ready? Experts say that the number of animal to human ‘species jumps’ is bound to increase as populations increase and the speed of travel between continents accelerates. Typical pandemic timelines no longer apply.¹ Pandemic H1N1 traveled the world in just weeks, as did SARS, despite major efforts to contain both outbreaks. The danger of emerging infectious disease to global health is compounded by the potential threat for malevolent bioengineering of existing pathogens and their deliberate dissemination.²

There is no panacea for emerging infectious disease, nor can we eliminate the threat of biowarfare. “Prevention is the best cure” and for that reason, vaccines may well be the answer. But which vaccine? How can we create a vaccine quickly enough to respond to an emergent pandemic? In response to the demand for safer, faster vaccines, the field of vaccine development is undergoing a complete transformation. In this article, we describe recent advances in vaccine design, delivery, and applications reported at the Fifth Vaccine Renaissance Conference (October 17–19, 2011; Providence RI).

The conference was organized by the Institute for Immunology and Informatics (iCubed) at the University of Rhode Island, and was the fifth Vaccine Renaissance Conference in seven years. This year, approximately 100 participants, ranging from academics to industry thought leaders attended lectures, networking events and hands‐on immunology and bioinformatics training.

One of the most important aspects of vaccine research is that it is highly “applied.” The relevance of vaccines to global health was quite evident in the mix of talks by invited speakers. Opening the conference, Dr. Mark Steinhoff, Division Director, Global Health Center (Cincinnati Children’s Hospital) described his ground-breaking research on the effects of vaccinating mothers to improve newborn health.¹ Influenza infection and disease during pregnancy affects the fetus in utero, lowering intrauterine growth. Dr Steinhoff also referenced literature describing the link between decreased intrauterine growth and adult obesity, coronary heart disease, diabetes and stroke.² Public health leaders and policy-makers would do well to pay heed to evidence that influenza immunization during pregnancy may have a significant impact on global health. Influenza vaccination during pregnancy provides protection for the mother, fetus, and young infant and is also safe, efficacious and cost effective.

In other good news, the economic landscape is changing for vaccine developers. Jeffrey Ulmer, PhD, Global Head of External Research at Novartis Vaccines and Diagnostics (Cambridge, MA) explained that the vaccine field was previously not well known for having blockbusters. The number of vaccine development companies has been limited to just a few players, and vaccine development has been characterized by lengthy development times. According to Ullmer, more biotech companies are engaging in vaccine development, and some vaccine blockbusters such as Prevnar^TM and Gardasil^TM now exist. Accelerated vaccine development processes have also been instituted to meet the demands of biodefense vaccine development. Dr Ulmer provided the case for using adjuvants to increase vaccine potency for the pandemic flu vaccines. He used the example of MF59, a squalene, oil and water adjuvant developed by Novartis which was shown to enhance H5N3 influenza vaccine effectiveness, provide broader recognition of H5N1 epitopes covering neutralizing regions of HA, induce unique B cell epitopes and enhance cell mediated immunity to H5N1. MF59 enhanced flu vaccine efficacy in 6 to 72 mo infants and appeared to act as an immune stimulus in the muscle promoting a local pro-inflammatory environment and stimulating the production of cytokines and chemokines.³

New means of accelerating the transfer of vaccine technology from bench to clinic will be an important driver of novel vaccine development. In his talk, Mark Poznansky, MD, PhD of Massachusetts General Hospital (MGH), described the Vaccine and Immunotherapy Center (VIC), a new collaborative effort between the MGH researchers, clinicians, and industry and academic partners to accelerate vaccine discovery. Dr Poznansky also described how enabling technologies such as new delivery vehicles will be key drivers of novel vaccine development. His ASAP or adjuvanted, self-assembling platform vaccine is one of several products being brought forward by VIC. ASAP is comprised of an immune-targeting construct (HSP70 to promote immune stimulatory function), avidin (for self-assembly), and a peptide or antigen, and biotin. The biotin and avidin form a tetrameric self-assembled construct containing four HSP70 molecules and four biotinylated-targeting molecules. Plans are underway to demonstrate proof of concept of this self-assembling vaccine technology for biodefense, in collaboration with EpiVax.

In addition to the ASAP technology, alternative vaccine delivery approaches were an exciting topic for discussion at the meeting. Hellen Amuguni, PhD, Tufts University Veterinary School, presented a low cost, temperature resistant mucosal vaccine delivery system using spores of the probiotic organism, Bacillus subtilis.⁴ In this system Bacillus has been bio-engineered to express recombinant antigens on the spore surface. The spores are then inactivated by heating before being administered intranasally or sublingually. Model antigens tested include tetanus, pertussis and rotavirus; similar to ASAP, the vaccine immunogenicity appears to be enhance by the size of the delivery vehicle.

Paulo Verardi, PhD, University of Connecticut, presented data on use of a vaccinia virus vector to deliver the fusion and hemagglutinin antigens of the Rinderpest virus, a pathogen of ruminants in Africa and Asia. This virus is one of the most recently eradicated viruses.⁵ The combination of these two antigens with the vaccinia vector provided robust sterilizing immunity against high doses of challenge virus. Edmund Gosselin, PhD, Albany Medical Centre, presented on the use of Fc targeted vaccines.⁶ His group has shown that mixture of specific antibody with antigen enhances vaccine mediated protection against tularemia, and that this enhanced protection requires the presence of the Fc component of the relevant antibodies as it is not seen with Fab fragments. The mechanism for the enhanced protection is not known but may be an effect of Fc receptor binding on enhanced T-cell IL-5 production, enhanced trafficking to draining lymph nodes or enhanced dendritic cell maturation. Gosselin has recently formed a company to exploit this effect to make an intranasal streptococcal pneumonia vaccine.

In addition to covering topics related to human pathogens and human vaccination, this year’s Fifth Vaccine Renaissance Conference was expanded to include the topics of “One Health” and “Animal Vaccines.” The One Health concept links human, animal and environmental health and is a collaborative effort of multiple disciplines to attain optimal health for people, animals and the environment. Dr Cyril Gay of the United States Department of Agriculture (USDA) provided an overview of One Health and its opportunities in biodefense and agriculture. The focus of the USDA in biodefense is to protect the global food supply, as this impacts world hunger, poverty and public health. Examples of threats to the global food supply are disease outbreaks and agroterrorism.^7,8 Examples of specific disease threats given were Highly-Pathogenic Avian Influenza, Foot-and Mouth Disease, Rift Valley Fever, Exotic Newcastle disease and Nipah and Hendra virus.⁹ As with human vaccine developers, veterinary researchers are looking toward innovations to improve vaccine supply such as the use of cell lines for vaccine production, novel expression systems, DNA vaccines and plant-based vaccines.

Following on the theme of One Health, Crystal Loving, PhD, USDA/ARS, presented on the issue of swine influenza, a problem not only for the swine themselves but also as a potential source of human influenza pandemics. Current swine influenza vaccines comprise 3–4 inactivated strains but there are increasingly too many circulating strains in swine for all variants to be covered. Another problem is that although inactivated vaccines may protect pigs against symptoms such as weight loss they do not protect against viral replication and reassortment, which increases the risk that the herds become a source of new viral strains for human pandemics. Dr Loving’s group at the USDA is therefore testing alternative vaccine technologies including DNA vaccines and Adenovirus vectors to see whether these might provide more robust protection against swine influenza. A trivalent DNA vaccine administered with 3 intramuscular doses had no effect on short-term virus shedding but did reduce late shedding by day 5–7 post challenge and induced both neutralizing antibody and CD4 T-cell responses. A replication deficient Ad5 vector given as a single dose at 4 weeks of age was less effective with no induction of neutralizing antibody although there was some reduction in virus on homologous challenge. A live attenuated temperature sensitive influenza virus (LAIV) vaccine administered as 2 intranasal doses induced only short-lived neutralizing antibody response but was effective in inducing T-cell responses.

Dr Loving also described the phenomena of vaccine-associated enhanced respiratory disease, which is sometimes seen when immunized swine receive a challenge with a heterologous influenza strain. Although the mechanism is not well understood, Dr Loving stated that this appears to be less of a problem with the LAIV and Ad5 vectored vaccines than the traditional inactivated vaccines. The timing of cytokine responses to influenza virus may be a factor.¹⁰

Veterinary pharmaceutical companies have significant resources to bring new animal vaccines to the market. Dr Randolph Seidler, Global Head of Research and Development in Animal Health at Boehringer Ingelheim Animal Health, GmbH, presented data on vaccine programs targeting swine diseases. Dr Seidler noted that in the past veterinary vaccines utilized inactivated or attenuated live vaccines but increasingly the move is toward subunit and vectored veterinary vaccines. As an example, a VLP-based PCV2 (Porcine Circovirus-2) vaccine (CircoFLEX) was developed that significantly reduced viral load and prevented disease-associated mortality in clinical studies. Pooled data from three clinical studies with n = 3000/group showed a mortality rate of ~65% in unimmunized compared with only 20% in immunized pigs. Boehringer Ingelheim is also developing new vaccines for Porcine Respiratory and Reproductive Syndrome Virus (PRRSV). Using Sequential Expression Library Immunization (SELI) (see ref. 11), cDNA clones were generated that sequentially represent all of the ORFs in the PRRSV genome. The ORF1a/b region was divided into 13 clones and clones representing the structural protein ORFs were also generated. The vaccinated animals showed a statistically significant reduction in lung lesions following challenge.

In addition to animal vaccines, new advances in fish vaccines were also presented. Erin Bromage, PhD from the University of Massachusetts at Dartmouth presented his work on unexpected B cell responses in trout following vaccination. Imports of fish and shellfish into the US create the 2nd largest trade deficit for natural resource products, and disease outbreak is the largest factor negatively impacting the success of aquaculture businesses. Designing effective vaccines for fish requires better understanding of fish immunology, which is still not well understood. Significant advances in fish immunology have been made in recent years for example: IgT appears to be analogous to IgA, IgM structure is modified following vaccination and fish IgD was discovered in 2011; a method for detecting T cells was also recently published.^7,8 Dr Bromage noted that only 14 vaccines are licensed for use in aquaculture and 10 of these are to the same target with the remaining 4 having questionable efficacy. In terms of basic fish biology, trout have neither bone marrow nor lymph nodes yet their kidney, which runs from the front gills to the anterior port, has immune tissue. IgM plasma cell distribution is primarily in the kidney, 60% in the head kidney and 30% in the posterior kidney, while 9% resides in the spleen and 5% in the blood. Studying plasma cell and antibody responses in rainbow trout following vaccination with TNP-KLH and FITC-CGG, it was discovered that the fish direct a significant proportion of their B cell responses to vaccination and that in the absence of creating new plasma cell capacity; new plasma cells displace resident plasma cells in order to survive. Dr Bromage surmises that the competitive displacement of old plasma cells secreting antibody for new plasma cells reacting to a current pathogen allows for the adaptation of humoral immunity in fish.

In one of the key presentations of the meeting, Nikolai Petrovsky, Flinders Medical Centre, Adelaide, Australia and Vaxine Pty Ltd, gave an overview of the rapid progress over the last decade in the field of immune-informatics, and highlighted how each of the important advances in the field had been captured in research papers published in Immunome Research (immunome-research.net), which continues to be the only scientific journal with an exclusive focus on immunoinformatics. He also highlighted contributions of immunoinformatics to advances in his own field of vaccine adjuvant research, where traditional trial and error approaches to adjuvant discovery have been superseded by targeted design approaches where adjuvants are designed from the ground up using immune-tailoring principles ensuring optimal immunogenicity. Using the example of Advax™, a polysaccharide adjuvant developed by Vaxine Pty Ltd, Petrovsky showed how knowledge of the polysaccharide crystal structure and its precise immunological effects as obtained through immuno-phenotyping had enabled optimization of adjuvant potency without compromising vaccine tolerability and safety.¹² This led to discussion of new high-throughput technologies for immune-phenotyping and vaccine characterization.

Determinant and measurement of the correlates of immunity is a critically important aspect of vaccine development. Sanchita Bhattachary, PhD, Stanford University, presented data on CYTOTOF, an exciting new multiparameter flow cytometry technique that combines traditional FACS with elemental labeled antibodies detected with mass spectroscopy (MS).¹³ Theoretically this will allow simultaneous characterization of up to 60 parameters, although at this stage it is working at the level of 30 parameters. Given the precise spectrum of elements on MS the problem of compensation seen with fluorochromes is not an issue with this system.

Similarly, Matthew Morrow, PhD, Inovio, presented data on the development of new cytotoxic T cell assays that are seen as important readouts for T cell directed DNA vaccines being developed by Inovio.¹⁴ These assays are directed at identification of granzyme B in the target cell population and are more specific than ELIspot assay. And finally, Barney Graham, Ph.D., NIH Vaccine Centre, presented data on how improved techniques for the characterization of neutralizing neutralizing antibodies of HIV and has shown that many of these antibodies recognize not just protein but also more complex glycan and lipid domains on the HIV surface.¹⁵ The next issue is to find how to enhance the production of these neutralizing HIV antibodies by optimizing parameters such as vaccine formulation, alternative immunization regimens, adjuvants, or time of antigen exposure. Given the large number of combination and permutations that will be involved in such multiparameter vaccine studies, informatics approaches will be critical in optimizing their design, just as new high-throughput immune-phenotyping methods will be critical to their execution. This provides strong evidence that immunoinformatics approaches will only increase in importance in future approaches to vaccine research and development.

A special session on Immunoinformatics with hands-on laboratory training was held during the conference; although all types of immunoinformatics tools were described, there are none that feature an integrated system such as iVAX (the academic version of the EpiMatrix vaccine design toolkit).¹⁶ Experts in computational biology instructed the attendees in the latest tools for vaccine design and the participants had the opportunity to use the tools.

In conclusion, the Vaccine Renaissance Conference continues to provide a solid bridge connecting those just entering the vaccine field with those already well established, and in particular has become a leading forum for discussion of cutting-edge concepts in the vaccine arena.