In recent years molecular biologists have developed an increasingly sophisticated understanding of the role of toll-like receptors (TLR) on various cells of the immune system and how ligand-binding of these receptors can either enhance or repress the activity of a particular cell type. As a result, various toll-like receptor ligands have been used as vaccine adjuvants with increasing frequency in recent years. 

The ability to modulate the immune response has been an important feature of vaccine adjuvants—specifically the ability of adjuvants to strengthen a relatively weak immune response to a particular antigen in a vaccine preparation. In recent years, molecular biologists have developed an increasingly sophisticated understanding of the role of toll-like receptors (TLR) on various cells of the immune system and how ligand-binding of these receptors can either enhance or repress the activity of a particular cell type. As a result, various TLR ligands have been used as vaccine adjuvants with increasing frequency in recent years. Indeed, a vaccine containing a TLR4 agonist (Cervarix) has recently been approved in the US and Europe. 

But the importance of immunomodulation is not limited to vaccine formulations. Augmenting the immune response can confer clinical benefits in a variety of diseases including cancer, while dampening the response can be important in therapeutics for autoimmune diseases. As such, the role of TLR agonists has expanded, and now the molecules can be found in a variety of non-vaccine therapeutics. 

In nature, TLR ligands are viral or bacterial components that bind TLRs and modulate the activity of the immune cell to which they preferentially bind. These days, molecules with TLR-binding activities can be synthesized and optimized for their binding affinities and immune-modulating function. 

 The first TLR7 agonist product, Aldara (imiquimod 5% cream), produced by 3M, was approved in 1997 for the topical treatment of external genital warts, actinic keratosis (a precancerous lesion of the skin), and basal cell carcinoma. Since then, 3M has synthesized well over 10,000 distinct molecules that act either as selective TLR7, selective TLR8, or as TLR7/8 agonists. These agonists have potential utility against a broad spectrum of diseases including a variety of dermatologic indications, atopic diseases like asthma and allergy, viral infections and cancers, in addition to a number of molecules for use as vaccine adjuvants with a variety of vaccines.  

A second generation topical TLR 7/8 agonist was recently licensed by 3M to a leading European dermatology company to treat a variety of skin conditions for use in Europe and Asia. Another TLR7 agonist that is more potent, more selective, and easier to formulate than imiquimod, 852A has been in Phase 1 studies and has shown a good safety profile in single-dose studies. It also is possible to use this topical cream for treatment of mucosal diseases, such as anogenital warts and human papilloma virus (HPV) infections of the vagina and cervix. Yet another selective TLR7 agonist, S-34240, can be used as a dermatologic product that can be sterilized. As such, it is being studied as a topical formulation for the treatment of diseases like post-surgical scarring and wrinkles. Currently, the molecule is in preclinical development. 

852A also has been developed in an injectable form for the treatment of cancer. The compound has been in Phase 2a studies to treat melanoma. In animal models, 852 has shown antitumor activity as a stand-alone agent and has demonstrated synergistic effects when given in combination with certain chemotherapeutic agents. Another follow-on compound, 854, activates immune cells through both TLR7 and TLR8 and is in preclinical development for the treatment of cancer. Because it has broader activation of immune cells than selective TLR7 agonists, it may have potential utility for cancers not treated by 852.  

TLR7 and TLR8 agonists have also been found to inhibit T helper type 2 (Th2) immune responses. Atopic diseases such as allergy and asthma appear to be because of overstimulation of Th2 responses, and therefore it is possible to treat allergy and asthma with a topically applied TLR7/8 agonist. Indeed, 3M scientists have shown that the TLR 7/8 agonist 854 is able to inhibit a number of activities associated with allergies and asthma. We have developed a nasal formulation of 854 for application to the nose to treat allergic rhinitis. This formulation has been evaluated in preclinical toxicology studies as well as rhinitis models and has shown good tolerability and efficacy in these models.   

As science continues to elucidate the complex controls governing immune responsiveness, we can expect to see an increasing role for TLR agonists as vaccine adjuvants and beyond.  

 is the business development director at 3M Drug Delivery Systems, St. Paul, MN, 651.733.5375, 

Articles

The role of TLR agonists has expanded, and now the molecules can be found in a variety of non-vaccine therapeutics.

Toll-Like Receptor Agonists: Not Just for Vaccines Anymore

Novartis recently built a plant for cell-based manufacture of influenza vaccine in Holly Springs, North Carolina. In this interview, 

, global head of technical operations for Novartis, talks about the company's plans for the new facility and new technologies for vaccine development and manufacturing.   

Novartis recently built a plant for cell-based manufacture of influenza vaccine in Holly Springs, North Carolina. What will be produced at the facility?

Novartis opened its Holly Springs, North Carolina facility in November 2009 in collaboration with the US Department of Health and Human Services (HHS). The opening of the Holly Springs manufacturing facility is part of our continued commitment to address urgent healthcare needs for our patients.  

The facility has been designed and constructed with the support of the US government to produce seasonal flu cell-culture vaccine, pre-pandemic [H5N1] and pandemic [H1N1] influenza vaccine. The facility will be ready to respond to an influenza pandemic as early as 2011 if licensed in an emergency. The plant is expected to be running at full scale and approved for commercial production in 2013. Additionally, the site will have a fill–finish facility capable of producing both flu and non-flu products. 

 Does Novartis also have an egg-based manufacturing program? If not, why not? If so, what are the challenges/limitations of producing vaccine in eggs? Are those challenges/limitations alleviated in cell-based systems? Are there challenges inherent in cell-based manufacture that are not challenges in egg-based systems?

Yes, Novartis uses both egg and cell-culture technology to produce vaccines in order to provide the greatest amount of vaccine. While egg-based technology is still the most widely used, cell-culture manufacturing offers several advantages over traditional egg-based manufacturing, such as the ability to more rapidly respond to time-critical situations, since there is no need for chicken eggs. Other advantages include more rapid and scalable manufacturing, enhanced consistency and reliability, and cell-culture-derived vaccines can be administered to patients who are allergic to eggs.

 Please give me a sense of the relative yields of egg- versus cell-based systems.

Novartis sets high standards for all its products and aims to achieve the highest yield possible using both egg-based and cell-based technologies.  As demonstrated from last year's A(H1N1) pandemic, yields are a biological process and cannot always be guaranteed. Producing high enough yields was one of the main challenges in developing the H1N1 vaccine because the volume of vaccine produced depends on how well the virus can replicate. However, cultivating viruses using a cell line offers the possibility of a more robust virus production and seed-strain development that more closely matches circulating viruses, which could potentially translate into a more immunogenic and effective response. 

 Please give me a sense of the start-up times for egg- versus cell-based paradigms. Scale-up?? 

A major advantage of using next-generation influenza manufacturing where eggs are no longer used is that the lead time to order and take delivery of eggs is eliminated. The Novartis proprietary cell line also allows for manufacturing flexibility. Because the lead time is eliminated, cell-culture-derived influenza vaccine can be produced more quickly and efficiently in emergency situations such as pandemics. For example, Novartis was able to produce the first monobulk vaccine for the A(H1N1) pandemic in only 60 days. 

 How do you purify your cell-based vaccines? How does this compare with egg-based purification paradigms?

The Novartis cell-derived influenza vaccines are purified in a way that is similar to that used for egg- based vaccines, though there are additional purification steps.  For example, host-cell proteins are removed during purification. In egg-based vaccines, some egg proteins remain in the vaccine. 

 Many people have said the cost of manufacturing vaccines in cells is prohibitive without government subsidies. The Holly Springs plant was built with a $485 million grant from the government. If the government had not bestowed this grant, would Novartis have built a cell-based manufacturing facility on its own?

New technologies are initially more expensive due to preliminary investment. Cell-based technology is an example of an innovation that required this type of investment. Once the technology is further developed and put into effect, the increased productivity makes the overall cost savings more apparent, which is why the partnership with the government was so important when building the facility. Additionally, cell-culture technology is beneficial because it provides a foundation for future biotechnology innovations. 

 If vaccine production is seasonal, how will the plant be used in the off season?

The facility in Holly Springs, NC, was designed to be capable of producing seasonal flu cell-culture vaccine, pre-pandemic vaccine, and 150 million doses of pandemic vaccine within 6 months of an influenza pandemic declaration. Additionally, the site will have a fill–finish facility capable of producing both flu and non-flu products.  

 Many companies are exploring the use of recombinant technologies for influenza vaccines. Does Novartis have such a program? If yes, please describe. If no, why not?

As a leader in innovative science and technology for vaccine development, Novartis Vaccines is not using only cell culture technology, but is also applying additional scientific breakthroughs such as adjuvants and reverse vaccinology to develop the most efficacious vaccines possible.  

Reverse vaccinology is a method by which viruses such as influenza are generated from segments of DNA. For prospective pandemic influenza vaccines, reverse vaccinology can be a useful technology because the process does not require manufacturers to work directly with potentially highly infectious pandemic strains, rather only segments of the virus's genome.  

 We understand that Novartis has a proprietary adjuvant. Could you describe what this adjuvant is and how it stimulates the immune system?

Novartis's proprietary adjuvant MF59 has an established safety profile. It is the only oil-in-water adjuvant supported by more than 12 years of post-marketing clinical safety data and more than 45 million doses of commercial use. The adjuvant has also been studied in randomized clinical trials and observational studies involving 124,000 individuals including children, adults, and elderly, and has been licensed for use in individuals 65 years of age and over in the seasonal influenza vaccine, FLUAD, since 1997 in the European Union. Benefits of vaccines produced with the MF59 adjuvant include established safety, immunogenicity, cross-reactivity, long-term boost-ability, T-cell immune memory, and antigen-sparing properties.  

This article was an online exclusive bonus to the 

Read more about vaccine development and manufacturing 

An Alternative Platform for Rapid Production of Effective Subunit Vaccines

Adjuvant activity can be greatly improved by appropriate formulation of cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG ODNs).

Sterilizing Filtration of Adjuvanted Vaccines: Ensuring Successful Filter Qualification

Filterability and bacterial retention must be verified very early in process development to ensure successful sterilizing filtration validation. 

Characterization of Aluminum Hydroxide Gel and Oil-in-Water Emulsion Formulations Containing CpG ODNs

			Adjuvant activity can be greatly improved by appropriate formulation of cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG ODNs).

Release and Stability Testing Programs for a Novel Virus-Like Particle Vaccine

Release testing involves both standard potency assays and unique assays (particle size, NA activity) developed to ensure the physical, chemical, and biological stability of this type of vaccine. 

The global head of technical operations at Novartis talks about new technologies for vaccine development and manufacturing.

Novartis's Influenza Vaccine Program

Despite intensive research since the viral pathogen was discovered some 25 years ago, not much progress has been reported on the development of a safe vaccine that protects against human immunodeficiency virus type 1. A vaccine approach that has been abandoned because its safety cannot be guaranteed is the single vaccine candidate that provides good protection in the macaque model, a live-attenuated variant of the simian immunodeficiency virus. The attenuated virus will cause a low-grade, but persistent infection that allows optimization of viral replication kinetics over time by spontaneous virus evolution, which may increase viral pathogenicity. In this article, we discuss innovative strategies to overcome this hurdle, including the generation of "single-cycle" viruses and a conditionally replicating HIV-1 variant. 

Vaccines that consist of a live-attenuated virus strain have proven to be very successful at inducing protective immunity against pathogenic viruses such as those causing smallpox, polio, and measles.

  Research on the development of a live-attenuated human immunodeficiency virus (HIV) vaccine has predominantly been performed in macaques that are infected with pathogenic simian immunodeficiency virus (SIV). Attenuation occurs by the deletion of several accessory functions from the viral genome, either individually or in combination.

  The majority of monkeys vaccinated with such deletion mutants of SIV can efficiently control replication of pathogenic challenge virus strains. However, the attenuated virus could revert to virulence and cause disease over time in vaccinated animals, especially in neonates.

  Similarly, some of the long-term nonprogressors of the Sydney Blood Bank Cohort, infected with a naturally attenuated HIV-1 variant with deletions in the nef and long terminal repeat (LTR) sequences eventually progressed to acquired immunodeficiency syndrome (AIDS).

  An HIV-1 Δ3 variant with deletions in the vpr, nef, and LTR sequences regained substantial replication capacity in long-term cell culture infections by acquiring compensatory changes in the viral genome.

  These results highlight the genetic instability and evolutionary capacity of attenuated SIV/HIV strains, which pose a serious safety risk for any future experimentation with live-attenuated HIV vaccines in humans. Novel strategies are needed to reconsider this vaccination approach because many other vaccine attempts have thus far failed to provide protection.

The Safety Issue of Live-Attenuated HIV-1 Variants

The major safety problem of live-attenuated HIV/SIV vaccine strains is related to the persistent replication and consequent evolution of the attenuated virus. In combination with the error-prone replication machinery of the virus, this ongoing low-level replication may eventually lead to the appearance of fitter and more pathogenic virus variants. To improve safety, the vaccine strain can be further attenuated through additional deletions or mutations in accessory genes or regulatory elements. This further reduces the pathogenic properties of the virus, but at the same time also reduces the vaccine efficacy.

  As an alternative strategy to prevent evolution toward a pathogenic variant, replication of the vaccine virus should be limited to the extent and time window that is required to provide full protection. For instance, virus replication can be stopped a few weeks after vaccination by administrating antiviral drugs.

  Although this is a good research strategy for macaque studies to address whether ongoing replication of the vaccine strain is needed for protection, application in humans seems problematic because long-term virus inhibition will require continuous drug administration, and the virus may develop drug resistance. Alternatively, a virus that can execute only a single round of replication can be used as a vaccine.

  However, because of the limited replication, such a single-cycle virus vaccine may be less potent for inducing protective immunity. We and others previously presented an alternative approach that uses a conditionally live HIV or SIV variant.

  We will discuss some of these approaches in this article. 

To reduce the risks associated with ongoing replication and evolution of live-attenuated SIV strains, single-cycle SIV variants (scSIV) have been designed. The initial scSIV variant was designed to use an artificial tRNA primer for reverse transcription that was exclusively present in the modified producer cell line, but not in other cells.

  In addition, attenuating deletions were introduced in the vif, vpr, vpx, and nef genes. After a single intravenous injection in rhesus macaques, peak viral RNA levels of 10

  copies/mL plasma were observed, indicating efficient expression of scSIV in the vaccinee. However, the vaccine doses used could not protect macaques from subsequent intravenous challenges with the pathogenic wild-type virus. A second approach for producing scSIV was based on Gag-Pol complementation of an SIV genome that is deficient for Pol expression as a result of a combination of mutations in the frameshift site that controls Pol translation.

  Four macaques were inoculated intravenously with three concentrated doses of scSIV, and viral loads peaked between 10

  On challenge, all animals became infected, but two of these animals were able to contain their viral loads below 2,000 RNA copies/mL as late as 35 weeks into the chronic phase of infection. These observations are encouraging and endorse future studies aimed at improving the protection.  

To improve the safety of a live-attenuated HIV-SIV vaccine strain, one must be able to shut-off virus replication once the vaccine has done its job. We introduced a genetic switch in the HIV-1 genome to control its replication. HIV gene expression and replication are naturally controlled by the viral Tat protein that binds to the 5' trans-acting responsive (TAR) region in the nascent RNA transcript to enhance transcription.

  For constructing a conditionally live HIV variant, this Tat–TAR regulatory mechanism was inactivated by mutation and functionally replaced by components of the doxycycline(dox)-inducible gene expression system (Tet-On system).

 gene-expression system is controlled by the rtTA protein, and binding of dox triggers a conformational switch that allows binding this protein to tet operator (tetO) elements and activation of transcription from the downstream positioned promoter. Thus, we introduced tetO elements in the LTR promoter and the rtTA gene in place of the nef gene. Transcription of this HIV-rtTA construct is activated by binding the dox-rtTA complex to the tetO-LTR promoter, and this virus replicates exclusively when dox is administered. After vaccination with this virus, replication can be temporarily activated by transient dox administration to the extent needed for induction of protective responses. The initial HIV-rtTA construct has been improved significantly by spontaneous virus evolution in prolonged cell culture infections.

  We have shown efficient and dox-dependent virus replication not only 

  An equivalent SIVmac variant was recently constructed that yielded promising results in vaccination tests in rhesus macaques (unpublished results). 

We realize that safety remains a major concern for drug-controlled virus variants. In fact, we constructed an HIV-1 variant that depends not only on dox for gene expression, but also on the T20 peptide for cell entry.

  T20 (Fuzeon) is a 36-mer peptide that mimics part of the HR2 domain of the envelope protein that is intrinsically involved in entry of the virus into the cell.

  We described the evolution of a T20-dependent HIV-1 variant in a patient on T20 therapy.

  This virus acquired two substitutions that created a hyperactive envelope protein. Further analysis revealed that the T20 peptide can rescue this hyperfusogenic protein by preventing the premature conformational switch, thus restoring virus infectivity and replication.

  Introducing these two mutations in HIV-rtTA resulted in a virus that replicates exclusively in the combined presence of dox and T20.

  Subsequent withdrawal of these inducers efficiently blocks viral replication and prevents ongoing evolution. 

We explored the possibility to use viruses based on HIV-1 strains that use CD4 and CXCR4 for cell entry as a therapeutic virus against malignancies such as T-lymphoblastic leukemia/lymphoma, NK leukemia, and some myeloid leukemias.

 The dox-controllable HIV-rtTA approach was combined with the design of a minimized HIV-1 variant for developing a virotherapy for cancer.

  This mini-HIV-rtTA variant lacks several nonessential genes and has lost the ability to replicate in normal primary cells, but this variant is still able to replicate in leukemic T-cell lines.

  This virus can efficiently and exclusively remove leukemic cells from a mixed culture with untransformed cells. In a therapeutic setting, the minimized virus can be used to target leukemic cells in the presence of dox. This will result in a self-limiting viral infection because the target cells are killed by the virus. Withdrawing dox provides an additional safety feature to block ongoing replication after the leukemic cells are removed.  

Dox-Controlled Virus for  Delivering RNAi Therapeutics 

We also explored the potential of the HIV-rtTA variant as a replicating vector for the efficient delivery of inhibitory gene cassettes that are based on the RNA interference (RNAi) mechanism. More specifically, we introduced an RNA polymerase III-driven short hairpin RNA (shRNA) cassette against wild-type HIV-1 sequences in the context of the dox-dependent virus.

  The shRNA targets the viral nef sequence, which is present in wild-type HIV-1 but not in the HIV-rtTA vector where the nef gene has been replaced by the rtTA gene. A spreading infection of this therapeutic HIV-rtTA-shRNAnef variant in HIV-susceptible cells can be controlled by transient dox treatment. Subsequent dox withdrawal generates cells that contain a silent integrated provirus with a constitutively active shRNAnef expression cassette. As a result, cells are harnessed with shRNAs that efficiently inhibit replication of wild-type HIV-1. This strategy seems particularly suitable for patients infected with a multidrug-resistant virus that can no longer be treated with the current antivirals. This HIV-rtTA-shRNAnef variant may allow interesting combinations of vaccination and RNAi-inhibition strategies. When used as a prophylactic vaccine, the RNAi cargo of this virus will protect all infected cells against a future exposure to HIV-1, thus boosting vaccine protection. When used as a therapeutic virus, the vaccine effect may boost the RNAi-mediated virus inhibition.  

Obvious safety concerns remain for developing replicating vectors based on the human pathogen HIV-1. One of the major concerns is that attenuated HIV-1 variants also cause a chronic infection. This fact, combined with the high mutation and recombination rate of HIV-1, may result in the generation of variants with altered replication characteristics over time. However, the dox-controlled HIV-rtTA variant will cause a latent infection on dox-withdrawal, with silent integrated proviruses that will less likely contribute to ongoing virus evolution because they are transcriptionally inactive. Another concern is that the vector may integrate near the 5' end of a proto-oncogene. In this position, the viral LTR-promoter may activate proto-oncogene expression, which could result in cell proliferation, and ultimately cause cancer. Such insertional oncogenesis occurred in 5 out of 20 patients who were treated with a gamma-retroviral vector,

  but the new generation lentiviral vectors were designed to be more safe, which is upheld in recent trials.

  The tetO-LTR promoter in our HIV-rtTA vectors is inactive on dox-withdrawal, which will strongly reduce the risk of activation of adjacent genes.  

We recently constructed a similar dox-dependent SIV variant, which is currently being used to study the efficacy and safety of a conditionally live virus vaccine against AIDS in macaques.

  This SIV variant may be a particularly attractive tool to study the correlates of immune protection on vaccination because the level and duration of replication can be controlled by dox administration. As a next step, the genetic stability and immunogenicity of the HIV-rtTA variant could be tested in mice with a humanized immune system.

  These results should indicate whether we can proceed on the risky path toward a live-attenuated HIV-1 vaccine. 

This research was funded by the Dutch AIDS Foundation (Aids Fonds Netherlands grants 7007, 2005–022, 2007–025), the Technology Foundation STW (applied science division of NWO and the technology program of the Ministry of Economic Affairs, The Netherlands), Zon-Medical Sciences (MW; VICI grant), and NWO-Chemical Sciences (CW; TOP grant). 

  is head of the Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands, 31.20 5663396,  

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Safety concerns remain for developing replicating vectors based on the pathogen human immunodeficiency virus type 1.

Evolution of Live-Attenuated HIV Vaccines

Virosomes are reconstituted viral envelopes that can serve as vaccines and as vehicles for cellular delivery of macromolecules. The prospect of drug delivery and targeting using virosomes is an interesting field of research and development. Because virosomes are biocompatible, biodegradable, nontoxic, and non-autoimmunogenic, attempts have been made to use them as vaccines or adjuvants as well as delivery systems for drugs, nucleic acids, or genes for therapeutic purposes. Influenza virus is the most common virus of choice. The success of virosomal drug delivery depends on the methods used to prepare the encapsulated bioactive materials and incorporate them into the virosomes, as are characterization and formulation of the finished preparation. Virosome technology could potentially be used to deliver peptides, nucleic acids or genes, and drugs like antibiotics, anticancer agents, and steroids.

Promising drugs are often discontinued during development because they cannot be suitably delivered to target cells, tissues, and organs. The new generation of therapeutics against cancer or neurodegenerative disorders require delivery systems that target drugs to specified cell types and host tissues by receptor-mediated uptake and controlled release. Virosomal technology presents a novel sophisticated delivery system to meet these challenges. 

Virosomes are reconstituted viral envelopes, including membrane lipids and viral spike glycoproteins, but devoid of viral genetic material. The external surface of the virosome resembles that of a virus particle, with spike proteins protruding from the membrane, but their interior compartment is empty. Virosomes were first prepared by Almeida et al., who inserted purified influenza spike proteins into preformed liposomes.

  Thereafter a range of viral envelopes have been reconstituted, including those of Sendai virus,

  Because virosomes display viral envelope glycoproteins, which, in their native conformation stimulate humoral responses, they are highly effective as vaccine antigens and adjuvants.

  Moreover, since the receptor-binding and membrane-fusion properties of the viral envelope glycoprotein can be preserved, virosomes can be used as transport vehicles for cellular delivery of biologically active macromolecules. In this article, we provide a brief overview of virosomal drug delivery. 

Overall, virosomes protect pharmaceutically active substances from proteolytic degradation and low pH within endosomes, allowing their contents to remain intact when they reach the cytoplasm. This is a major advantage of virosomal carrier systems over other drug-delivery vehicles, including liposomal and proteoliposomal carrier systems. 

Virosomal technology is approved by the FDA for use in humans, and has a high safety profile 

virosomes are biodegradable, biocompatible, and non-toxic

broadly applicable with almost all important drugs (anticancer drugs, proteins, peptides, nucleic acids, antibiotics, fungicides)

enables drug delivery into the cytoplasm of target cell 

promotes fusion activity in the endolysosomal pathway 

Virosomes are spherical unilamellar vesicles with a mean diameter of around 150 nm. Influenza virus is most commonly used for virosome production. Virosomes cannot replicate but are pure fusion-active vesicles. In contrast   to   liposomes,  virosomes   contain   functional   viral   envelope glycoproteins:  influenza virus hemagglutinin (HA) and neuraminidase (NA) are intercalated within the phospholipid bilayer membrane (Figure 1). Further characteristics of virosomes depend on the choice of bilayer components. Virosomes can be optimized for maximal incorporation of the drug, or for the best physiological effect by modifying the content or type of membrane lipids used. It is even possible to generate carriers for antisense-oligonucleotides or other genetic molecules, depending on whether positively or negatively loaded phospholipids are incorporated into the membrane. Various ligands, such as cytokines, peptides, and monoclonal antibodies (MAbs) can be incorporated into the virosome and displayed on the virosomal surface. Even tumor-specific monoclonal antibody fragments (Fab) can be linked to virosomes to direct the carrier to selected tumor cells.

Figure 1. Virosomes are reconstituted infl uenza virus envelopes devoid of inner core and genetic information 

Liposomes have been considered promising vehicles for targeting and delivery of biologically active molecules to living cells both 

. However, liposomes have little potential to fuse with cells and thus, generally fail to provide appreciable delivery of encapsulated molecules to the cell cytoplasm. In contrast, virosomes   contain   functional   viral   envelope glycoproteins with receptor-binding and membrane-fusion properties that enable the cellular delivery of encapsulated molecules.

Virosomes have unique fusion properties because of the presence of influenza HA in their membranes. HA not only confers structural stability and homogeneity to virosomal formulations, but it also significantly contributes to the fusion activity of virosomes. Virosomal HA promotes binding at the target cell surface followed by receptor-mediated endocytosis. The acidic environment of the endosome triggers HA-mediated membrane fusion, and the therapeutically active substance escapes from the endosome into the cytoplasm of the target cell. Thus, virosomal  HA  significantly  enhances  cytosolic  delivery.  Overall, virosomes protect pharmaceutically active substances from proteolytic degradation and low pH within the endosomes before they reach the cytoplasm. This is a major advantage of the virosomal carrier system over liposomal and proteoliposomal carrier systems, which provide less protection for therapeutic macromolecules from harsh compartmental microenvironments.

To prepare virosomes, a viral membrane-fusion protein such as HA—the generally preferred fusion protein for virosomes—is either purified from the corresponding virus or produced recombinantly. The success of virosomes as a vaccine or delivery vehicle requires that reconstituted membrane proteins retain their immunogenic properties as well as their receptor-binding and membrane-fusion activities. This involves functional reconstitution of influenza virus membranes, which is based on solubilizing viral membranes by nondenaturing detergents. Influenza virus envelopes incorporated with HA can be solubilized with nonionic detergents having a relatively low critical micellar concentration (CMC). Octaethylene glycol mono (n-dodecyl) ether (C12E8) is the most commonly used detergent. Triton X-100 is a frequently used alternative detergent. Other nonionic detergents also can be used.

Following solubilization, the viral nucleocapsid, which contains the endogenous viral genes, is removed by ultracentrifugation. The viral membranes are reconstituted when C12E8 is removed by adsorption onto a hydrophobic resin. Virosomes produced by this method fuse in a pH-dependent manner similar to native influenza virus.  

The C12E8 method has certain inherent drawbacks. This method involves batch processing, often in open systems. This is a challenging situation for industrial processing, particularly to maintain sterility. Furthermore the compounds to be encapsulated within the virosomes could be adsorbed or inactivated by the hydrophobic resin. It is also difficult to remove low-CMC detergents like C12E8 for solubilization from the system. Detergent removal by dialysis can circumvent these complications.

Dialysis requires the use of detergents with relatively high CMCs, such as N-octyl- -D-glucopyranoside (octyl glucoside), that can effectively solubilize influenza virus envelopes. However, fusogenic virosomes are not readily prepared by subsequent removal of the octyl glucoside detergent. During dialysis, the HA concentrates primarily in lipid-poor aggregates with a very limited aqueous space, while the viral lipid is recovered in protein-poor vesicles. Although these vesicles exhibit some HA-mediated membrane fusion activity, only a small fraction of the HA is recovered in these vesicles. Researchers are in pursuit of novel detergents and detergent-like compounds that can be almost completely removed by dialysis. These will be crucial for refining an effective dialysis procedure to reconstitute influenza virus membranes for industrial purposes.

Other lipids also can be added to the membranes during preparation. These lipids include cholesterol and phospholipids such as phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and phosphatidylserine. Cationic lipids also are added to concentrate nucleic acids in the virosomes or to facilitate virosome-mediated cellular delivery of nucleic acids or genes. These include, DOTAP: (N-[1-(2,3-dioleoyloxy) propyl] - N,N,N-trimethylammonium chloride), DODAC: (N,N-dioleyl-N,N, dimethylammonium chloride), stearylamine, etc. DODAC is the preferred cationic lipid for complexing nucleic acids to the virosome to ensure cellular delivery of nucleic acids. Concentrations of DODAC in the range of 25–45% are particularly good to ensure cellular delivery of nucleic acids.

Additional components can be added to the virosomes to target them to specific cell types. For example, virosomes can be conjugated to MAbs that bind cellular epitopes present on the surfaces of specific cell types. 

 Virosome preparation should generally result in a relatively uniform protein-to-lipid ratio. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) can confirm the presence of HA protein in the virosomes.

Negative-stain electron microscopy can generally be used to determine the ultrastructure and size of virosomes. The staining solutions should preferably be of neutral pH, to avoid acid-induced conformational changes of HA.

 Generally virosomes exhibit pH-dependent membrane fusion activity similar to native influenza virus. Virosomal fusion with biological or artificial target membranes can be visualized with a fluorescent resonance energy transfer assay (RET).

  Alternatively, fusion can be assessed in vitro with an excimer assay using pyrene-labeled lipids, where  the decrease of surface density of the pyrene-phosphatidylcholine-label on fusion with an unlabeled membrane corresponds to a reduction of excimer fluorescence.

Fusion activity also can be indirectly monitored by determining hemolytic activity, which  corresponds closely to fusion activity and exhibits a pH dependence identical with that of fusion.

Bioactive drug compounds can be entrapped in the aqueous interior of the virosome or in the lipid membrane of the virosome for facilitated entry of the compounds into the cells.

Virosomes are particularly useful for delivering nucleic acids or genes. These compounds are delivered into the host cell cytoplasm on fusion of the virosome with the endosome or plasma membrane.

  Nucleic acids or genes encoding a naturally occurring protein can be introduced into host cells and can be expressed, provided that the expression cassette contains the proper cis-acting regulatory elements.

Drugs or nucleic acids can be incorporated into the virosome at the time of virosome preparation. The bioactive compound is typically added to the lipid–HA-containing solution following removal of the nucleocapsid. Alternatively, the bioactive compound is initially incorporated into a liposome, which is then fused with a virosome containing two hemagglutinins with different pH thresholds to form a virosome–liposome hybrid.

Proteins also can be delivered to cells via virosome. For example, the gelonin subunit A of diphtheria toxin and ovalbumin have also been successfully delivered by virosome to target cells.

  Virosomes carrying peptides derived from the influenza nucleoprotein or intact ovalbumin induced strong cytotoxic T lymphocyte responses, which suggests that the encapsulated peptides and proteins gained access to the cytoplasm.

Ideally one would like to be able to target drug delivery to selected tissues. One can tailor virosomes to targets by incorporating specific molecules (e.g., Fab fragments and ligands) into the virosome's composition. The feasibility of targeted delivery of anticancer drugs by means of virosomal carrier has been demonstrated recently by two independent approaches.  In one,  a  MAb cross-linked to the surface of virosomes mediated specific targeting of the virosomal carrier containing an anticancer drug (e.g., doxorubicin) to human cancer  cells. MAbs can bind specifically to cancer-related antigens, providing a means to target systemically administered virosomes to cancerous tissues. Alternatively, ligands that bind surface receptors on the target cells also can be bound to the virosomes to achieve targeted drug delivery. Tumors of mice treated with targeted drug-loaded virosomes failed to grow, and mortality of these animals was significantly reduced. These positive results will definitely open a new field of applications for virosomal technology.

Several formulations have been reported. Generally, virosomes are suspended in buffered saline (135–150 mM NaCl), but other suitable vehicles also exist. These compositions should be sterilized by conventional liposomal sterilization techniques, such as membrane filtration. The formulation also generally contains auxiliary substances as required to simulate physiological conditions, such as buffering agents and isotonicity adjusting agents (sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride).

  The concentration of virosomes used in the vehicle ranges from 20–200 mg/mL. These concentrations are varied to optimize treatment with different virosome components or for particular purposes.

The virosomes are administered in a variety of parenteral routes, including intravenous, intramuscular, subcutaneous, intra-arterial, and inhalable delivery. In addition, virosomes can be administered topically, orally, or transdermally. The virosomes also can be incorporated into implantable devices for long-term release.

Virosomes present novel drug-delivery vehicles with distinct advantages over liposomes.

BioPharm International-01-02-2011

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