Synthetic Virus-like Particles Research Articles

An SI-traceable reference material for virus-like particles

SummaryA reference material for virus-like particles traceable to the International System of Units (Système International d'Unités – the SI) is reported. The material addresses the need for developing reference standards to benchmark virus-like gene delivery systems and help harmonize measurement approaches for characterization and testing. The material is a major component of synthetic polypeptide virus-like particles produced by the state-of-the-art synthetic and analytical chemistry methods used to generate gene delivery systems. The purity profile of the material is evaluated to the highest metrological order demonstrating traceability to the SI. The material adds to the emerging toolkit of reference standards for quantitative biology.

Epicutaneous immunization using synthetic virus-like particles efficiently boosts protective immunity to respiratory syncytial virus

Despite the substantial health and economic burden caused by RSV-associated illness, no vaccine is available. The sole licensed treatment (palivizumab), composed of a monoclonal neutralizing antibody, blocks the fusion of the virus to the host cell but does not prevent infection. The development of a safe and efficacious RSV vaccine is therefore a priority, but also a considerable challenge, and new innovative strategies are warranted. Most of the adult population encounter regular RSV infections and can elicit a robust neutralizing antibody response, but unfortunately it wanes over time and reinfections during subsequent seasons are common. One approach to protect the mother and young infant from RSV infection is to administer a vaccine capable of boosting preexisting RSV immunity during pregnancy, which would provide protection to the fetus through passive transfer of maternal antibodies, thus preventing primary RSV infection in newborns during their first months of life. Here, we describe the preclinical evaluation of an epicutaneous RSV vaccine booster that combines epicutaneous patches as a delivery platform and a Synthetic Virus-Like Particles (SVLP)-based vaccine displaying multiple RSV F-protein site II (FsII, palivizumab epitope) mimetic as antigen (V-306). We demonstrated in mice that epicutaneous immunization with V-306 efficiently boosts preexisting immunity induced by the homologous V-306 administered subcutaneously. This boosting was characterized by a significant increase in F- and FsII-specific antibodies capable of competing with palivizumab for its target antigen and neutralize RSV. More importantly, epicutaneous booster immunization with V-306 significantly decreased lung viral replication in experimental mice after intranasal RSV challenge, without inducing enhanced RSV disease. In conclusion, an epicutaneous booster vaccine based on V-306 is safe and efficacious in enhancing RSV preexisting immunity in mice. This needle-free vaccine candidate would be potentially suited as a booster vaccine for vulnerable populations such as young infants via pregnant women, and the elderly.

Programmable Membrane-Mediated Attachment of Synthetic Virus-like Nanoparticles on Artificial Protocells for Enhanced Immunogenicity

The programming of protocells to implement and control the functions of living cells through activated membrane receptor signaling remains a challenge for the development of protoliving technologies. Here, we report a synthetic protocell capable of inducing high levels of macrophage activation in vitro. An immunogenic protocell comprising hyaluronic acid (HA)-polymer membrane decorated with synthetic virus-like particles derived from the functionalization of core-shell polymer nanoparticles with a Toll-like receptor agonist (Pam3SK4) and cell/protocell membrane-binding lectin (WGA) is fabricated. While the non-decorated HA-based protocells and functionalized SVLPs alone initiate moderate levels of macrophage activation, co-presentation of WGA and Pam3SK4 on the protocell membrane results in hyperactivation of the phagocytes via a synergistic multi-receptor process. This work offers opportunities for developing chemically programmable soft microscale agents capable of eliciting cellular signal transduction and genetic outputs and provides a step toward implementing future therapeutic strategies based on cognate interactions at the cell-protocell interface.

Chitosan in Non-Viral Gene Delivery: Role of Structure, Characterization Methods, and Insights in Cancer and Rare Diseases Therapies.

Non-viral gene delivery vectors have lagged far behind viral ones in the current pipeline of clinical trials of gene therapy nanomedicines. Even when non-viral nanovectors pose less safety risks than do viruses, their efficacy is much lower. Since the early studies to deliver pDNA, chitosan has been regarded as a highly attractive biopolymer to deliver nucleic acids intracellularly and induce a transgenic response resulting in either upregulation of protein expression (for pDNA, mRNA) or its downregulation (for siRNA or microRNA). This is explained as the consequence of a multi-step process involving condensation of nucleic acids, protection against degradation, stabilization in physiological conditions, cellular internalization, release from the endolysosome (“proton sponge” effect), unpacking and enabling the trafficking of pDNA to the nucleus or the siRNA to the RNA interference silencing complex (RISC). Given the multiple steps and complexity involved in the gene transfection process, there is a dearth of understanding of the role of chitosan’s structural features (Mw and degree of acetylation, DA%) on each step that dictates the net transfection efficiency and its kinetics. The use of fully characterized chitosan samples along with the utilization of complementary biophysical and biological techniques is key to bridging this gap of knowledge and identifying the optimal chitosans for delivering a specific gene. Other aspects such as cell type and administration route are also at play. At the same time, the role of chitosan structural features on the morphology, size and surface composition of synthetic virus-like particles has barely been addressed. The ongoing revolution brought about by the recent discovery of CRISPR-Cas9 technology will undoubtedly be a game changer in this field in the short term. In the field of rare diseases, gene therapy is perhaps where the greatest potential lies and we anticipate that chitosans will be key players in the translation of research to the clinic.

Dendritic Cell Sensing of Hydrophobic Di- and Triacylated Lipopeptides Self-Assembled within Synthetic Virus-like Particles.

Dendritic cells (DCs) play critical roles in developing immune defenses. One important aspect is interaction with pathogen-associated molecular patterns (PAMPs)/danger-associated molecular patterns, including di- and triacylated lipopeptides. Isolated or synthetic lipopeptides are potent vaccine adjuvants, interacting with cell surface TLR2 heterodimers. In contrast, deep embedment within bacteria cell walls would impair lipopeptide interaction with cell surface TLR2, requiring degradation for PAMP recognition. Accordingly, DC processing in the absence of surface TLR2 ligation was defined using synthetic virus-like particles (SVLPs) carrying hydrophobic TLR2 PAMPs within di- and triacylated lipopeptide cores (P2Cys-SVLPs and P3Cys-SVLPs) compared with SVLPs lacking immunomodulatory lipopeptides. DCs rapidly and efficiently internalized SVLPs, which was dominated by slow endocytic processing via macropinocytosis, although some caveolar endocytosis was implicated. This delivered SVLPs primarily into macropinosomes often interacting with EEA-1+ early endosomes. Although endoplasmic reticulum association was occasionally noted, association with recycling/sorting structures was not observed. Involvement of LysoTracker+ structures slowly increased with time, with SVLPs present in such structures ultimately dominating. Only SVLPs carrying di- and triacylated lipopeptide cores induced DC activation and maturation independently of surface TLR2 ligation. Intracellular recognition of SVLP TLR2 ligands was confirmed by observing SVLPs' association with internal TLR2, which had similar kinetics to SVLP association with LysoTracker. This related to inflammatory cytokine induction by SVLP+ DCs, with adaptive immune response activation ex vivo/in vivo. Importantly, particular DCs, not monocytes, recognized intracellular exposure of the TLR2 PAMPs carried by di- and triacylated SVLP cores, which indicates subset-distinct recognition of functional internal TLR2 ligands. Thus, vaccines carrying hydrophobic TLR2 ligands would interact with particular DCs for efficient induction of specific immunity in the absence of additional adjuvant.

Protein Epitope Mimetics: From New Antibiotics to Supramolecular Synthetic Vaccines.

Protein epitope mimetics provide powerful tools to study biomolecular recognition in many areas of chemical biology. They may also provide access to new biologically active molecules and potentially to new classes of drug and vaccine candidates. Here we highlight approaches for the design of folded, structurally defined epitope mimetics, by incorporating backbone and side chains of hot residues onto a stable constrained scaffold. Using robust synthetic methods, the structural, biological, and physical properties of epitope mimetics can be optimized, by variation of both side chain and backbone chemistry. To illustrate the potential of protein epitope mimetics in medicinal chemistry and biotechnology, we present studies in two areas of infectology; the discovery of new antibiotics targeting essential outer membrane (OM) proteins in Gram-negative bacteria and the design of supramolecular synthetic vaccines. The discovery of new antibiotics with novel mechanisms of action, in particular to combat infections caused by Gram-negative pathogens, represents a major challenge in medicinal chemistry. We were inspired by naturally occurring cationic antimicrobial peptides to design structurally related peptidomimetics and to optimize their antimicrobial properties through library synthesis and screening. Through these efforts, we could show that antimicrobial β-hairpin mimetics may have structures and properties that facilitate interactions with essential bacterial β-barrel OM proteins. One recently discovered family of antimicrobial peptidomimetics targets the β-barrel protein LptD in Pseudomonas spp. This protein plays a key role in lipopolysaccaride (LPS) transport to the cell surface during OM biogenesis. Through a highly selective interaction with LptD, the peptidomimetic blocks LPS transport, resulting in nanomolar antimicrobial activity against the important human pathogen P. aeruginosa. Epitope mimetics may also have great potential in the field of vaccinology, where structural information on complexes between neutralizing antibodies and their cognate epitopes can be taken as a starting point for B cell epitope mimetic design. In order to generate potent immune responses, an effective method of delivering epitope mimetics to relevant cells and tissues in the immune system is also required. For this, engineered synthetic nanoparticles (synthetic virus-like particles, SVLPs) prepared using supramolecular chemistry can be designed with optimal surface properties for efficient dendritic cell-mediated delivery of folded B-cell and linear T-cell epitopes, along with ligands for pattern recognition receptors, into lymphoid tissues. In this way, multivalent display of the epitope mimetics occurs over the surface of the nanoparticle, suitable for cross-linking B cell receptors. In this highly immunogenic format, strong epitope-specific humoral immune responses can be elicited that target infections caused by pathogenic microorganisms. Other potential applications of epitope mimetics in next-generation therapeutics are also discussed.

Synthetic virus-like particles prepared via protein corona formation enable effective vaccination in an avian model of coronavirus infection.

The ongoing battle against current and rising viral infectious threats has prompted increasing effort in the development of vaccine technology. A major thrust in vaccine research focuses on developing formulations with virus-like features towards enhancing antigen presentation and immune processing. Herein, a facile approach to formulate synthetic virus-like particles (sVLPs) is demonstrated by exploiting the phenomenon of protein corona formation induced by the high-energy surfaces of synthetic nanoparticles. Using an avian coronavirus spike protein as a model antigen, sVLPs were prepared by incubating 100 nm gold nanoparticles in a solution containing an optimized concentration of viral proteins. Following removal of free proteins, antigen-laden particles were recovered and showed morphological semblance to natural viral particles under nanoparticle tracking analysis and transmission electron microscopy. As compared to inoculation with free proteins, vaccination with the sVLPs showed enhanced lymphatic antigen delivery, stronger antibody titers, increased splenic T-cell response, and reduced infection-associated symptoms in an avian model of coronavirus infection. Comparison to a commercial whole inactivated virus vaccine also showed evidence of superior antiviral protection by the sVLPs. The study demonstrates a simple yet robust method in bridging viral antigens with synthetic nanoparticles for improved vaccine application; it has practical implications in the management of human viral infections as well as in animal agriculture.

A Synthetic Virus-Like Particle Streptococcal Vaccine Candidate Using B-Cell Epitopes from the Proline-Rich Region of Pneumococcal Surface Protein A.

Alternatives to the well-established capsular polysaccharide-based vaccines against Streptococcus pneumoniae that circumvent limitations arising from limited serotype coverage and the emergence of resistance due to capsule switching (serotype replacement) are being widely pursued. Much attention is now focused on the development of recombinant subunit vaccines based on highly conserved pneumococcal surface proteins and virulence factors. A further step might involve focusing the host humoral immune response onto protective protein epitopes using as immunogens structurally optimized epitope mimetics. One approach to deliver such epitope mimetics to the immune system is through the use of synthetic virus-like particles (SVLPs). SVLPs are made from synthetic coiled-coil lipopeptides that are designed to spontaneously self-assemble into 20–30 nm diameter nanoparticles in aqueous buffer. Multivalent display of epitope mimetics on the surface of SVLPs generates highly immunogenic nanoparticles that elicit strong epitope-specific humoral immune responses without the need for external adjuvants. Here, we set out to demonstrate that this approach can yield vaccine candidates able to elicit a protective immune response, using epitopes derived from the proline-rich region of pneumococcal surface protein A (PspA). These streptococcal SVLP-based vaccine candidates are shown to elicit strong humoral immune responses in mice. Following active immunization and challenge with lethal doses of streptococcus, SVLP-based immunogens are able to elicit significant protection in mice. Furthermore, a mimetic-specific monoclonal antibody is shown to mediate partial protection upon passive immunization. The results show that SVLPs combined with synthetic epitope mimetics may have potential for the development of an effective vaccine against Streptococcus pneumoniae.

Analysis of purified wild type and mutant adenovirus particles by SILAC based quantitative proteomics.

We used SILAC (stable isotope labelling of amino acids in cell culture) and high-throughput quantitative MS mass spectrometry to analyse the protein composition of highly purified WT wild type adenoviruses, mutant adenoviruses lacking an internal protein component (protein V) and recombinant adenoviruses of the type commonly used in gene therapy, including one virus that had been used in a clinical trial. We found that the viral protein abundance and composition were consistent across all types of virus examined except for the virus lacking protein V, which also had reduced amounts of another viral core protein, protein VII. In all the samples analysed we found no evidence of consistent packaging or contamination with cellular proteins. We believe this technique is a powerful method to analyse the protein composition of this important gene therapy vector and genetically engineered or synthetic virus-like particles. The raw data have been deposited at proteomexchange, identifer PXD001120.

Unravelling capsid transformations

The interactions between a virus capsid and its cargo are essential for viral infection as well as in the design of synthetic virus-like particles. Now a combination of analytical techniques has unravelled key steps in the transformation of a model virus and the release of its RNA cargo.

Abstract LB-3: Fully synthetic self-assembling tumor-targeted virus-like nanoparticles .

Abstract Virus particles have evolved as highly effective delivery systems. They are able to deliver whole proteins and large nucleic acid molecules to certain types of cells. Genetically engineered virus-like particles (VLPs) have been explored as drug delivery and imaging agents. However, generation of fully synthetic VLPs has not been achieved. We have found that properly derivatized synthetic analogs of transmembrane domains not only inhibit the function of the corresponding receptor, but can self-assemble into virus-like particles with high precision (Proc. Natl. Acad. Sci. U. S. A 108: 9798 (2011). The described particles have intrinsic biological activity of the peptide they are composed of and fuse with cell spontaneously. Particles composed of CXCR4 antagonist fuse spontaneously with cell membrane, inhibit CXCR4 function in vitro and CXCR4-mediated metastasis in vivo. However, the fusion is non-selective. We have now developed the first fully synthetic VLPs that fuse with cells like real viruses, in receptor-mediated manner. Prevention of non-selective fusion was achieved by derivatization of self-assembling peptides with polyethylene glycol. Addition of ligands recognizing receptors over expressed on prostate tumor cells allowed for the particles that fuse selectively with receptor-positive cells. Particles targeting gastrin-releasing peptide receptor (GRPR), luteinizing hormone-releasing hormone (LHRH) receptor and prostate-specific membrane antigen (PSMA) are remarkably stable, more than 99 % homogeneous in size and undergo receptor-mediated fusion with tumor cells expressing corresponding receptor. Whole- animal fluorescence tomography of nude mice injected with fluorescent nanoparticles targeted to PSMA and GRPR revealed that particles rapidly accumulate in tumors and allow for tumor detection as early as two hours after the injection. Development of fully synthetic VLPs establishes a foundation for generation of delivery systems with investigator-defined biophysical properties and targeting ability for a wide range of biomedical applications. Tumor-targeted self-assembling particles represent a new class of agents with dual and triple biological activity for the therapy and detection of tumors. Citation Format: Nadya Tarasova, Yuhong Chen, Marzena Dyba, Sergey G. Tarasov. Fully synthetic self-assembling tumor-targeted virus-like nanoparticles . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-3. doi:10.1158/1538-7445.AM2013-LB-3

Max Bergmann lecture protein epitope mimetics in the age of structural vaccinology.

This review highlights the growing importance of protein epitope mimetics in the discovery of new biologically active molecules and their potential applications in drug and vaccine research. The focus is on folded β-hairpin mimetics, which are designed to mimic β-hairpin motifs in biologically important peptides and proteins. An ever-growing number of protein crystal structures reveal how β-hairpin motifs often play key roles in protein–protein and protein–nucleic acid interactions. This review illustrates how using protein structures as a starting point for small-molecule mimetic design can provide novel ligands as protein–protein interaction inhibitors, as protease inhibitors, and as ligands for chemokine receptors and folded RNA targets, as well as novel antibiotics to combat the growing health threat posed by the emergence of antibiotic-resistant bacteria. The β-hairpin antibiotics are shown to target a β-barrel outer membrane protein (LptD) in Pseudomonas sp., which is essential for the biogenesis of the outer cell membrane. Another exciting prospect is that protein epitope mimetics will be of increasing importance in synthetic vaccine design, in the emerging field of structural vaccinology. Crystal structures of protective antibodies bound to their pathogen-derived epitopes provide an ideal starting point for the design of synthetic epitope mimetics. The mimetics can be delivered to the immune system in a highly immunogenic format on the surface of synthetic virus-like particles. The scientific challenges in molecular design remain great, but the potential significance of success in this area is even greater. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

Phototriggered cargo release from virus-like assemblies

There has been tremendous progress towards the development of responsive polymers that are programmed to respond to an external stimulus such as light, pH and temperature. The unique combination of molecular packaging followed by slow, controlled release of molecular cargo is of particular importance for self-healing materials and the controlled release of drugs. While much focus and progress remains centred around synthetic carriers, viruses and virus-like particles can be considered ideal cargo carriers as they are intrinsically designed to package, protect and deliver nucleic acid cargo to host cells. Here, we report the encapsulation of a stimuli-responsive self-immolative polymer within virus-like assemblies of Cowpea Chlorotic Mottle Virus. Upon photo-irradiation, the self-immolative polymer undergoes a head-to-tail depolymerization into its monomeric subunits, resulting in the slow release of the molecular cargo. We propose that the liberated monomers are small enough to diffuse through the pores of the virus capsid shell and offer an alternative strategy for the controlled loading and unloading of the molecular cargo using viruses as cargo carriers.

Synthetic Virus-Like Particles Target Dendritic Cell Lipid Rafts for Rapid Endocytosis Primarily but Not Exclusively by Macropinocytosis

DC employ several endocytic routes for processing antigens, driving forward adaptive immunity. Recent advances in synthetic biology have created small (20–30 nm) virus-like particles based on lipopeptides containing a virus-derived coiled coil sequence coupled to synthetic B- and T-cell epitope mimetics. These self-assembling SVLP efficiently induce adaptive immunity without requirement for adjuvant. We hypothesized that the characteristics of DC interaction with SVLP would elaborate on the roles of cell membrane and intracellular compartments in the handling of a virus-like entity known for its efficacy as a vaccine. DC rapidly bind SVLP within min, co-localised with CTB and CD9, but not caveolin-1. In contrast, internalisation is a relatively slow process, delivering SVLP into the cell periphery where they are maintained for a number of hrs in association with microtubules. Although there is early association with clathrin, this is no longer seen after 10 min. Association with EEA-1+ early endosomes is also early, but proteolytic processing appears slow, the SVLP-vesicles remaining peripheral. Association with transferrin occurs rarely, and only in the periphery, possibly signifying translocation of some SVLP for delivery to B-lymphocytes. Most SVLP co-localise with high molecular weight dextran. Uptake of both is impaired with mature DC, but there remains a residual uptake of SVLP. These results imply that DC use multiple endocytic routes for SVLP uptake, dominated by caveolin-independent, lipid raft-mediated macropinocytosis. With most SVLP-containing vesicles being retained in the periphery, not always interacting with early endosomes, this relates to slow proteolytic degradation and antigen retention by DC. The present characterization allows for a definition of how DC handle virus-like particles showing efficacious immunogenicity, elements valuable for novel vaccine design in the future.

Synthetic Virus‐Like Particles and Conformationally Constrained Peptidomimetics in Vaccine Design

Conformationally constrained peptidomimetics could be of great value in the design of vaccines targeting protective epitopes on viral and bacterial pathogens. But the poor immunogenicity of small synthetic molecules represents a serious obstacle for their use in vaccine development. Here, we show how a constrained epitope mimetic can be rendered highly immunogenic through multivalent display on the surface of synthetic virus-like nanoparticles. The target epitope is the V3 loop from the gp120 glycoprotein of HIV-1 bound to the neutralizing antibody F425-B4e8. The antibody-bound V3 loop adopts a β-hairpin conformation, which is effectively stabilized by transplantation onto a D-Pro-L-Pro template. The resulting mimetic after coupling to synthetic virus-like particles elicited antibodies in rabbits that recognized recombinant gp120. The elicited antibodies also blocked infection by the neutralization sensitive tier-1 strain MN of HIV-1, as well as engineered viruses with the V1V2 loop deleted; this result is consistent with screening of V3 by the V1V2 loop in intact trimeric viral gp120 spikes. The results provide new insights into HIV-1 vaccine design based on the V3 loop, and illustrate how knowledge from structural biology can be exploited for the design of constrained epitope mimetics, which can be delivered to the immune system by using a highly immunogenic synthetic nanoparticle delivery system.

Human Papilloma Virus Vaccines: Implications for use in developing world

Cervical cancer may be substantially prevented by human papillomavirus (HPV) vaccination. The two most common oncogenic HPV types causing 70% of all cervical cancers are represented in the vaccines by synthetic virus-like particles to the L1 protein of HPV 16 and 18. The virus-like particles and adjuvant systems promote long-term antibody response. Invasive cervical cancer (ICC) is the second most common cancer among women worldwide. Approximately 85% of the disease burden is seen in women in developing nations. India with its highest share of global burden of cervical cancer has to implement a population based cervical cancer control program to reduce the number of deaths. Development of effective vaccines against human papillomavirus, the necessary cause of cervical cancer, has introduced a fresh lease of life to the cervical cancer control strategies. Vaccination programs targeted to a large age range of women will achieve cervical cancer reductions several decades from now.

Synthetic Viruslike Particles and Hybrid Constructs Based on Lipopeptide Self‐Assembly

Synthetic Viruslike Particles and Hybrid Constructs Based on Lipopeptide Self‐Assembly

Synthetic Viruslike Particles for Targeted Gene Delivery to αvβ3 Integrin-Presenting Endothelial Cells

Progress in the design of gene delivery systems is of utmost importance for cancer gene therapy since several physiological and intracellular barriers remain. We previously developed a technology for condensing a single gene into a single and stable globular nanometric system. In this manuscript, we have decorated the nanometric particles with cyclic RGD ligands in order to target endothelial cells. The potential of these artificial viruses as targeted gene delivery vehicles is demonstrated in vitro with alpha(v)beta(3) integrin-expressing primary endothelial cells.

Engineering Clustered Ligand Binding Into Nonviral Vectors: αvβ3 Targeting as an Example

The development of techniques to efficiently deliver genes using nonviral approaches can broaden the application of gene delivery in medical applications without the safety concerns associated with viral vectors. Here, we designed a clustered integrin-binding platform to enhance the efficiency and targetability of nonviral gene transfer to HeLa cells with low and high densities of alpha(v)beta(3) integrin receptors. Arg-Gly-Asp (RGD) nanoclusters were formed using gold nanoparticles functionalized with RGD peptides and used to modify the surface of DNA/poly(ethylene imine) (PEI) polyplexes. DNA/PEI polyplexes with attached RGD nanoclusters resulted in either 5.4- or 35-fold increase in gene transfer efficiency over unmodified polyplexes for HeLa cells with low- or high-integrin surface density, respectively. The transfection efficiency obtained with the commercially available vector jetPEI-RGD was used for comparison as a vector without clustered binding. JetPEI-RGD exhibited a 1.2-fold enhancement compared to unmodified jetPEI in cells with high densities of alpha(v)beta(3) integrin receptors. The data presented here emphasize the importance of the RGD conformational arrangement on the surface of the polyplex to achieve efficient targeting and gene transfer, and provide an approach to introduce clustering to a wide variety of nanoparticles for gene delivery.

Prophylactic human papillomavirus vaccines to prevent cervical cancer: review of the Phase II and III trials

Cervical cancer may be substantially prevented by human papillomavirus (HPV) vaccination. The two most common oncogenic HPV types causing 70% of all cervical cancers are represented in the vaccines by synthetic virus-like particles to the L1 protein of HPV 16 and 18. The virus-like particles and adjuvant systems promote long-term antibody response. Phase II trials indicated vaccine efficacy against type-specific infection, initial vaccine immunogenicity and the tolerability of initial vaccination. Although designed to be preliminary evidence of disease prevention, the Phase II trials have become the sentinel beacon of longer-term efficacy for infection and disease outcomes, immunogenicity and safety after at least 5 years of follow-up. Both vaccines were approved for licensure after corroborative favorable Phase III trial data on efficacy, immunogenicity and safety. Vaccination programs targeted to a large age range of women will achieve cervical cancer reductions several decades from now.