Gary T. Jennings

Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns

Researchers working on the development of vaccines face an inherent dilemma: to maximize immunogenicity without compromising safety and tolerability. Early vaccines often induced long-lived protective immune responses, but tolerability was a major problem. Newer vaccines have very few side effects but can be of limited immunogenicity. One way to tackle this problem is to design vaccines that have all the properties of pathogens with the exception of causing disease. Key features of pathogens that can be mimicked by vaccine delivery systems are their size, shape and surface molecule organization. In addition, pathogen-associated molecular patterns can be used to induce innate immune responses that promote adaptive immunity. In this Review, we discuss the approaches currently being used to optimize the delivery of antigens and enhance vaccine efficacy.

The coming of age of virus-like particle vaccines.

Abstract Virus-like particles are supra-molecular assemblages, usually icosahedral or rod-like structures. They incorporate key immunologic features of viruses which include repetitive surfaces, particulate structures and induction of innate immunity through activation of pathogen-associated molecular-pattern recognition receptors. They carry no replicative genetic information and can be produced recombinantly in large scale. Virus-like particles thus represent a safe and effective vaccine platform for inducing potent B- and T-cell responses. In addition to being effective vaccines against the corresponding virus from which they are derived, virus-like particles can also be used to present foreign epitopes to the immune system. This can be achieved by genetic fusion or chemical conjugation. This technological innovation has greatly broadened the scope of their use, from immunizing against microbial pathogens to immunotherapy for chronic diseases. Towards this end, virus-like particles have been used to induce autoantibodies to disease-associated self-molecules involved in chronic diseases, such as hypertension and Alzheimers disease. The recognition of the potent immunogenicity and commercial potential for virus-like particles has greatly accelerated research and development activities. During the last decade, two prophylactic virus-like particle vaccines have been registered for human use, while another 12 vaccines entered clinical development.

A molecular assembly system that renders antigens of choice highly repetitive for induction of protective B cell responses

Virus like particles (VLPs) are known to induce potent B cell responses in the absence of adjuvants. Moreover, epitope-specific antibody responses may be induced by VLPs that contain peptides inserted in their immunodominant regions. However, due to steric problems, the size of the peptides capable of being incorporated into VLPs while still permitting capsid assembly, is rather limited. While peptides genetically fused to either the N- or C-terminus of VLPs present fewer assembly problems, the immune responses obtained against such epitopes are often limited, most likely because the epitopes are not optimally exposed. In addition, such particles may be less stable in vivo. Here, we show that peptides and proteins engineered to contain a free cys can be chemically coupled to VLPs formed from the hepatitis B core antigen (HBcAg) containing a lys in the immuno-dominant region. By using this approach steric hindrance of capsid assembly is abrogated. Peptides or protein coupled to VLPs in an oriented fashion are shown to induce strong and protective B cell responses even against self-epitopes in the absence of adjuvants. This molecular assembly system may be used to induce strong B cell responses against most antigens.

A therapeutic vaccine for nicotine dependence: preclinical efficacy, and phase I safety and immunogenicity

Nicotine is the principal addictive component in tobacco, and following uptake acts in the central nervous system. The smoking‐cessation efforts of most smokers fail because a single slip often delivers sufficient nicotine to the brain to reinstate the drug‐seeking behaviour. Blocking nicotine from entering the brain by induction of specific antibodies may be an effective means to prevent such relapses. The hapten nicotine was coupled to virus‐like particles (VLP) formed by the coat protein of the bacteriophage Qb. In preclinical experiments, this Nicotine–Qb VLP (NicQb) vaccine induced strong antibody responses. After intravenous nicotine challenge, vaccinated mice exhibited strongly reduced nicotine levels in the brain compared with control mice. In a phase I study, 32 healthy non‐smokers were immunized with NicQb. The vaccine was safe and well‐tolerated. All volunteers who received NicQb showed nicotine‐specific IgM antibodies at day 7 and nicotine‐specific IgG antibodies at day 14. Antibody levels could be boosted by a second injection or the addition of Alum as an adjuvant and the antibodies had a high affinity for nicotine. These data suggest that antibodies induced by NicQb may prevent relapses by sequestering nicotine in the blood of immunized smokers.

Effect of immunisation against angiotensin II with CYT006-AngQb on ambulatory blood pressure: a double-blind, randomised, placebo-controlled phase IIa study

BACKGROUND Hypertension can be controlled adequately with existing drugs such as angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers. Nevertheless, treatment success is often restricted by patients not adhering to treatment. Immunisation against angiotensin II could solve this problem. We investigated the safety and efficacy of CYT006-AngQb-a vaccine based on a virus-like particle-that targets angiotensin II to reduce ambulatory blood pressure. METHODS In this multicentre, double-blind, randomised, placebo-controlled phase IIa trial, 72 patients with mild-to-moderate hypertension were randomly assigned with a computer-generated randomisation list to receive subcutaneous injections of either 100 mug CYT006-AngQb (n=24), 300 mug CYT006-AngQb (24), or placebo (24), at weeks 0, 4, and 12. 24-h ambulatory blood pressure was measured before treatment and at week 14. The primary outcomes were safety and tolerability. Analyses were done by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00500786. FINDINGS Two patients in the 100 mug group, three in the 300 mug group, and none in the placebo group discontinued study treatment. All patients were included in safety analyses; efficacy analyses did not include the five dropouts, for whom no data were available at week 14. Five serious adverse events were reported (two in the 100 mug group, two in the 300 mug group, and one in the placebo group); none were deemed to be treatment related. Most side-effects were mild, transient reactions at the injection site. Mild, transient influenza-like symptoms were seen in three patients in the 100 mug group, seven in the 300 mug group, and none in the placebo group. In the 300 mug group, there was a reduction from baseline in mean ambulatory daytime blood pressure at week 14 by -9.0/-4.0 mm Hg compared with placebo (p=0.015 for systolic and 0.064 for diastolic). The 300 mug dose reduced the early morning blood-pressure surge compared with placebo (change at 0800 h -25/-13 mm Hg; p<0.0001 for systolic, p=0.0035 for diastolic). INTERPRETATION Immunisation with CYT006-AngQb was associated with no serious adverse events; most observed adverse events were consistent with local or systemic responses similar to those seen with other vaccines. The 300 mug dose reduced blood pressure in patients with mild-to-moderate hypertension during the daytime, especially in the early morning. FUNDING Cytos Biotechnology AG.

Vaccination against IL‐17 suppresses autoimmune arthritis and encephalomyelitis

Interleukin 17 is a T cell‐derived cytokine that induces the release of pro‐inflammatory mediators in a wide range of cell types. Recently, a subset of IL‐17‐producing T helper cells (Th17) distinct from Th1 and Th2 cells has been described, which constitutes a new T cell polarization state. Aberrant Th17 responses and overexpression of IL‐17 have been implicated in a number of autoimmune disorders including rheumatoid arthritis and multiple sclerosis. Molecules blocking IL‐17 such as IL‐17‐specific monoclonal antibodies have proved to be effective in ameliorating disease in animal models. Hitherto, active immunization targeting IL‐17 is an untried approach. Herein we explore the potential of neutralizing IL‐17 by active immunization using virus‐like particles conjugated with recombinant IL‐17 (IL‐17‐VLP). Immunization with IL‐17‐VLP induced high levels of anti‐IL‐17 antibodies thereby overcoming natural tolerance, even in the absence of added adjuvant. Mice immunized with IL‐17‐VLP had lower incidence of disease, slower progression to disease and reduced scores of disease severity in both collagen‐induced arthritis and experimental autoimmune encephalomyelitis. Active immunization against IL‐17 therefore represents a novel therapeutic approach for the treatment of chronic inflammatory diseases.

Versatile Virus-Like Particle Carrier for Epitope Based Vaccines

Background Recombinant proteins and in particular single domains or peptides are often poorly immunogenic unless conjugated to a carrier protein. Virus-like-particles are a very efficient means to confer high immunogenicity to antigens. We report here the development of virus-like-particles (VLPs) derived from the RNA bacteriophage AP205 for epitope-based vaccines. Methodology/Principal Findings Peptides of angiotensin II, S.typhi outer membrane protein (D2), CXCR4 receptor, HIV1 Nef, gonadotropin releasing hormone (GnRH), Influenza A M2-protein were fused to either N- or C-terminus of AP205 coat protein. The A205-peptide fusions assembled into VLPs, and peptides displayed on the VLP were highly immunogenic in mice. GnRH fused to the C-terminus of AP205 induced a strong antibody response that inhibited GnRH function in vivo. Exposure of the M2-protein peptide at the N-terminus of AP205 resulted in a strong M2-specific antibody response upon immunization, protecting 100% of mice from a lethal influenza infection. Conclusions/Significance AP205 VLPs are therefore a very efficient and new vaccine system, suitable for complex and long epitopes, of up to at least 55 amino acid residues in length. AP205 VLPs confer a high immunogenicity to displayed epitopes, as shown by inhibition of endogenous GnRH and protective immunity against influenza infection.

The Second-Generation Active Aβ Immunotherapy CAD106 Reduces Amyloid Accumulation in APP Transgenic Mice While Minimizing Potential Side Effects

Immunization against amyloid-β (Aβ) can reduce amyloid accumulation in vivo and is considered a potential therapeutic approach for Alzheimers disease. However, it has been associated with meningoencephalitis thought to be mediated by inflammatory T-cells. With the aim of producing an immunogenic vaccine without this side effect, we designed CAD106 comprising Aβ1–6 coupled to the virus-like particle Qβ. Immunization with this vaccine did not activate Aβ-specific T-cells. In APP transgenic mice, CAD106 induced efficacious Aβ antibody titers of different IgG subclasses mainly recognizing the Aβ3–6 epitope. CAD106 reduced brain amyloid accumulation in two APP transgenic mouse lines. Plaque number was a more sensitive readout than plaque area, followed by Aβ42 and Aβ40 levels. Studies with very strong overall amyloid reduction showed an increase in vascular Aβ, which atypically was nonfibrillar. The efficacy of Aβ immunotherapy depended on the Aβ levels and thus differed between animal models, brain regions, and stage of amyloid deposition. Therefore, animal studies may not quantitatively predict the effect in human Alzheimers disease. Our studies provided no evidence for increased microhemorrhages or inflammatory reactions in amyloid-containing brain. In rhesus monkeys, CAD106 induced a similar antibody response as in mice. The antibodies stained amyloid deposits on tissue sections of mouse and human brain but did not label cellular structures containing APP. They reacted with Aβ monomers and oligomers and blocked Aβ toxicity in cell culture. We conclude that CAD106 immunization is suited to interfere with Aβ aggregation and its downstream detrimental effects.

A Virus-Like Particle-Based Vaccine Selectively Targeting Soluble TNF-α Protects from Arthritis without Inducing Reactivation of Latent Tuberculosis

Neutralization of the proinflammatory cytokine TNF-α by mAbs or soluble receptors represents an effective treatment for chronic inflammatory disorders such as rheumatoid arthritis, psoriasis, or Crohn’s disease. In this study, we describe a novel active immunization approach against TNF-α, which results in the induction of high titers of therapeutically active autoantibodies. Immunization of mice with virus-like particles of the bacteriophage Qβ covalently linked to either the entire soluble TNF-α protein (Qβ-C-TNF1–156) or a 20-aa peptide derived from its N terminus (Qβ-C-TNF4–23) yielded specific Abs, which protected from clinical signs of inflammation in a murine model of rheumatoid arthritis. Whereas mice immunized with Qβ-C-TNF1–156 showed increased susceptibility to Listeria monocytogenes infection and enhanced reactivation of latent Mycobacterium tuberculosis, mice immunized with Qβ-C-TNF4–23 were not immunocompromised with respect to infection with these pathogens. This difference was attributed to recognition of both transmembrane and soluble TNF-α by Abs elicited by Qβ-C-TNF1–156, and a selective recognition of only soluble TNF-α by Abs raised by Qβ-C-TNF4–23. Thus, by specifically targeting soluble TNF-α, Qβ-C-TNF4–23 immunization has the potential to become an effective and safe therapy against inflammatory disorders, which might overcome the risk of opportunistic infections associated with the currently available TNF-α antagonists.

Active immunization with IL‐1 displayed on virus‐like particles protects from autoimmune arthritis

IL‐1 is an important mediator of inflammation and a major cause of tissue damage in rheumatoid arthritis (RA). Therapeutic administration of recombinant IL‐1 receptor antagonist (IL‐1Ra) is efficacious in reducing clinical symptoms of disease, but suffers from several drawbacks, including the need for frequent administrations of large amounts. Here, we show that immunization of mice with either IL‐1α or IL‐1β chemically cross‐linked to virus‐like particles (VLP) of the bacteriophage Qβ elicited a rapid and long‐lasting autoantibody response. The induced Ab efficiently neutralized the binding of the respective IL‐1 molecules to their receptors in vitro and their pro‐inflammatory activities in vivo. In the collagen‐induced arthritis model, both vaccines strongly protected mice from inflammation and degradation of bone and cartilage. Moreover, immunization with either vaccine showed superior efficacy than daily administrations of high amounts of IL‐1Ra. In the T and B cell‐independent collagen Ab transfer model, immunization with the IL‐1β vaccine strongly protected from arthritis, whereas immunization with the IL‐1α vaccine had no effect. Our results suggest that active immunization with IL‐1α, and especially IL‐1β conjugated to Qβ VLP, might become an efficacious and cost‐effective new treatment option for RA and other systemic IL‐1‐dependent inflammatory disorders.