Where are we going?

Vaccines in the 21st century

Margaret A Liu, vice president. 

Vaccines Research and Gene Therapy, Chiron Corporation, 4560 Horton Street, MS 4.6, Emmeryville, CA 94608, USA

margaret-liu{at}cc.chiron.com

Vaccination against infectious diseases has literally transformed the planet. It has succeeded in the elimination of smallpox and the near elimination of polio as scourges of humankind. This kind of success, as well as the great increase in understanding of immunology and the development of new technologies, has increased the hope that new vaccines will target other diseases. Indeed, expectations have been raised so much that vaccines are now being developed not just to prevent infectious diseases but to cure them. In addition, some conditions have now been shown to be related to infectious diseasesfor example, human papillomavirus infection has been implicated in cervical cancer and Helicobacter pylori in gastritis, peptic ulcer disease, and stomach cancerand vaccines should help to prevent or treat these diseases. Other clinical diseases or conditions are also now considered within the purview of vaccines. These include cancers (even those without a known infectious aetiological component), allergies, autoimmune diseases such as diabetes, metabolic diseases such as hyperlipidaemias that lead to atherosclerosis and coronary artery disease, and even addictions such as cocaine and nicotine dependencies.

An examination of the recent advances in immunology and vaccinology provides insights into future vaccine developments. Great strides have been made in understanding how the body distinguishes self from non-selfthat is, how the immune system recognises a pathogen as being foreign and hence a target for killing. This is likewise important for understanding why the immune response sometimes becomes too active, resulting in allergies or autoimmune disease. By understanding which cells become activated and knowing what the triggers are, we can try intentionally to stimulate, alter, or suppress the responses.

The elucidation of how immune system cells process and then recognise and respond to antigens derived from pathogens has prompted the development of new technologies to try to mimic or even improve on these responses. Thus, whereas most existing vaccines were made simply and successfully by administering the pathogen in a weakened, killed, or dissected form, new approaches are much more "surgical." That is, they attempt to induce specific types of immunity (particularly cellular responses or particular classes of antibody) and against selected structures, rather than against the whole virus or bacterium or those parts that could easily be purified. In addition, the ability to sequence the genome of whole organisms has provided a new tool for identifying which proteins from a pathogen to include in a vaccine. Additionally, more effort is being applied to generating specific regional immunity at the site most relevant to prevent either infection (for example, mucosal surfaces) or disease.

Possibly the most intriguing revelations have been in the field of cellular immune responsesspecifically, insights into how T cells recognise their targets and what occurs to the antigen in the target cells to make it into a form that can activate T cells. It has been recognised that a number of pathogens, for which we do not have vaccines, as well as immunotherapies for cancer, would require the generation of these T cells to prevent, limit, or treat disease. Earlier vaccine technologies may not be effective for generating these responses or may be considered problematic from a safety perspective for a given disease. For example, though live attenuated virus vaccines are quite effective for a number of diseases, they may in some instances revert to wild type virulence (for example, polio). Thus, using an attenuated HIV strain as a vaccine for HIV is considered by many to be too risky. Armed with an increased understanding of what is required to generate T cell response, "vaccinologists" have focused on finding ways to deliver antigen specifically to activate these T cells.

	Technologies

Top Technologies Methods of delivery Clinical targets for vaccines Social factors References

Thus, insights into disease aetiology and pathogenesis and the development of new technologies that enable vaccinologists to capitalise on new immunological insights have been critical for this effort in new vaccines. Specific examples of new vaccine developments are given below as illustrations of various issues.

Recombinant DNA technology
The first example of a technology that has had an impact on vaccine development is recombinant DNA technology. Cloning has enabled the production of an effective hepatitis B vaccine that does not require the manufacture or purification of the intact or infectious virus. Other molecular biology tools have made it possible to identify which proteins are conserved among different strains of a virus or bacteria, or which are responsible for virulence of pathogens or oncogenesis in tumours. These proteins may be good candidates for vaccines.

(Credit: CHIRON) View larger version (92K): [in this window] [in a new window]   Child being administered FluMist

Molecular attenuation of pathogens
Recombinant DNA technology has further resulted in the ability to manipulate genes so that, rather than relying on blind serial growth of viruses or bacteria in an effort to weaken them for use as live vaccines, pathogens can be attenuated or altered molecularly so that they are unable to replicate effectively or cause disease. For example, BCG is a live mycobacterium used in many countries as a vaccine against primary tuberculosis, but its effectiveness varies widely. This is partly because of the different derivations of different vaccine strains. Certain mutants, such as auxotrophic mutants, have been made through manipulation of the Mycobacterium tuberculosis genome, which results in the production of strains that cannot replicate unless they are provided with particular nutrients that would not be present in a human.¹ Likewise, salmonella and shigella strains are being attenuated molecularly so that they will still infect via the mucosal route, but will not be able to replicate.² Thus, by eliminating precisely and molecularly the pathogenicity from the pathogen, new vaccines may be made safer and more effective.

Vectors
Furthermore, a gene from a pathogen can be inserted into an innocuous agent so that the antigen can now be delivered in the form of a gene via an unrelated and non-pathogenic virus or bacterium. This technology is being used for attenuated versions of BCG, salmonella, and shigella. Vaccinia and a bird version of pox virus are also being evaluated as vectors for delivering the gene of a heterologous virus (notably HIV) in continuing clinical trials.³

A new type of vector, which should enter clinical trials in the next few years for HIV and hepatitis C virus, is based on the class of positive stranded RNA viruses known as alphaviruses.⁴ These vectors provide several attractive features. They replicate by making multiple copies of mRNA encoding the structural proteins. Thus, when used as a heterologous expression vector, they cause the cell to make large quantities of the protein antigen quite rapidly. The replicon particle seems to be a normal viral particle on the outside, and hence can use some of the strategies that the wild type virus uses for infecting cells efficiently. However, its genome has been altered so that the cell can produce the antigen for the vaccine but not the replica structural proteins, and thus the cell cannot make any progeny virus. These particles, when made from certain strains of alphavirus, have the capability of targeting specialised immune cells called dendritic cells, which function to present antigen to stimulate cytotoxic T lymphocytes.

View larger version (87K): [in this window]

DNA vaccines
DNA vaccines, which are simply a bacterial plasmid encoding an antigen, are an even simpler form of gene vaccine that has been developed recently. These DNA vaccines have entered into clinical trials for a number of diseases such as HIV, malaria, and cancer.⁵ DNA vaccines have generated considerable interest because they are simple and there is no need to make an intact virus or bacterium. Efforts to increase their potency are ongoing.

View larger version (75K): [in this window]

	Methods of delivery

Top Technologies Methods of delivery Clinical targets for vaccines Social factors References

Live poliovirus vaccines administered orally are the greatest current example of a practitioner and user friendly, economically feasible, and clinically effective vaccine. Given all of these attributes and the fact that most pathogens enter the host via a mucosal surface, vaccine development efforts have focused heavily on finding ways of delivering many types of antigens to the mucosa with and without the use of a live organism.

A new vaccine for influenza is based on the development of influenza strains that grow well only in the cooler temperatures of the nasal passages, but do not replicate in the warmer lower respiratory tract. This vaccine has been shown to be effective in children and adults after intranasal administration. ^{6 7} Non-living systems utilising adjuvants (such as molecularly attenuated toxins from bacteria) are also being developed. An example of this is LTK63, a heat labile toxin of Escherichia coli which has been made recombinantly with specific mutations to eliminate its toxic ability. ^{8 9} It has none of the toxic activity of the native protein, yet is a powerful adjuvant when coadministered with antigens in preclinical studies.

Oral delivery is an even more attractive route of administration from the patient's perspective, and several approaches are being developed. One approach has been to encapsulate the vaccine in such a way that it survives the gastric passage and reaches the intestine, which is rich in immune cells. In addition, some of the live attenuated organisms (such as shigella and salmonella described above) that normally invade the intestinal epithelium are being tested both as vaccines and as delivery vectors for antigens from other pathogens.^10-13

An approach that has great appeal in relation to the potential ease of both production and delivery is using transgenic edible plants to make vaccines. For example, genes encoding antigens have been cloned into potato plants. The potato makes the protein, and hence by eating the potato, animalsand more recently humanshave been shown to develop immune responses. ^{14 15} The drawback of using potatoes is that they are usually eaten cooked, and this may denature a protein antigen. Thus, bananas are considered the ideal food base for a vaccine. Of course, fundamental questions still need to be addressed, since intestinal administration of some antigens in certain forms has proved to be an effective means of generating antigen specific tolerance. But the appeal of making an edible vaccine, especially one that infants would enjoy receiving, is great. Another advantage is that agricultural production will probably be less costly than biological manufacturing.

One of the functions of skin has been presumed to be protection from direct entry of entities (whether pathogens or compounds) into the body. Yet, the transdermal patch has been an efficacious and patient friendly means of delivering systemic drugs. Vaccines of the future may be delivered through the skin by patches or other needleless devices.¹⁶ These approaches would not only provide an advantage from the recipient's perspective but may be able to harness the wealth of antigen presenting cells in the skin.

Adjuvants
Adjuvants are compounds that increase the immunogenicity of a given vaccine. Alum has long been the only compound approved for human use, but a vaccine with an adjuvant known as MF59 was licensed recently.¹⁷ In the future, MF59 and other compounds still in development (for example, LTK63, see above) ^{8 9 18} will be used even more widely to reduce the number of immunisations or increase the potency in people who otherwise would not respond.

(Credit: NYAS/JEFF CAMPBELL) View larger version (34K): [in this window] [in a new window]   FluMist being sprayed

	Clinical targets for vaccines

Top Technologies Methods of delivery Clinical targets for vaccines Social factors References

Infectious diseases
There are still many diseases for which we need vaccines ... or better vaccines. The misuse of antibiotics has resulted in the increasing development of antibiotic resistant strains of bacteria. Furthermore, as useful and effective as antiviral drugs have been, their limitations have been painfully obvious for agents such as hepatitis C virus and HIV, where the side effects or rigours of the treatment regimen, or both, reduce the number of people who are treated. Other problems, such as the cost of treatment and the limited population of patients who respond, also underline the continued need for vaccines. In addition to the future development of successful vaccines for pathogens such as HIV, hepatitis C virus, malaria, and herpes simplex virus, new vaccines will be made which will be more effective against pathogens such as M tuberculosis, for which a vaccine already exists. In addition, though all vaccines to date have been for prophylaxis, vaccines will be developed to treat infectious diseases. For example, since the immune response is clearly capable of clearing infection for a number of pathogens that can also cause chronic infection, it is logical to try to stimulate such a therapeutic immune response in individuals who have not succeeded to clear infection on their own. Examples of such vaccines under evaluation include ones for hepatitis B virus, hepatitis C virus, HIV, tuberculosis, and human papillomavirus infections.

Cancer
Cancer gene therapy in the future will probably include the gene based delivery not only of anti-angiogenic molecules but also of tumour specific antigens in conjunction with cytokines. Therapeutic cancer vaccines illustrate how the concept of vaccines has widened to encompass non-infectious diseases and treatment. Furthermore, cancer vaccines illustrate the increasing blur between the areas of gene therapy and vaccines. The vectors used for so called gene vaccines, which consist of either a DNA plasmid or of a viral vector only, are also being used for some gene therapies.

Allergies
Efforts to harness the immune response and turn it in another direction are also being used to develop vaccines for allergies. For example, a type 2 T helper response (the production of cytokines such as interleukin 4 and interleukin 10, which are necessary for making antibody responses) versus a type 1 T helper response (cytokines such as interleukin 2 and  interferon, which are particularly relevant for cellular responses) to particular antigens has been implicated in the pathogenesis of certain allergies and autoimmune states. Thus, vaccines are being developed to specifically manipulate the antigen specific response to be predominantly a type 1 T helper response.

Other conditions
While gene therapists work towards curing diabetes through gene therapy, vaccinologists are striving to prevent or treat this and other autoimmune diseases by efforts to induce antigen specific tolerance and by altering the immunological milieu to change the type of immune response.

View larger version (97K): [in this window]

	Social factors

Top Technologies Methods of delivery Clinical targets for vaccines Social factors References

Social factors have also had an impact on vaccine development. For example, more effort is being made to eliminate the need for syringe injections, in an effort to make vaccines both more accessible in parts of the world and more acceptable from the patient's perspective. Likewise, the knowledge that each year millions of children worldwide die from infectious diseases for which vaccines exist, has spurred efforts to make more stable vaccines that do not need refrigeration or devices for delivery. And finally, the very success of vaccines at eliminating untold morbidity and mortality and the increased understanding of immune responses have raised expectations to desire prevention or cure of a myriad of diseases in addition to infectious diseases. The proved value of vaccines, coupled with powerful new technologies, promise that they will have an increasing role in efforts to improve the health of the denizens, both human and animal, of the planet.

Competing interests: MAL is an employee of Chiron and former employee of Merck; both companies are in the vaccine business.

	References

Top Technologies Methods of delivery Clinical targets for vaccines Social factors References

1.	Guleria I, Teitelbaum R, McAdam RA, Kalpana G, Jacobs WRJ, Bloom BR. Auxotrophic vaccines for tuberculosis. Nature Med 1996; 2: 334-337[Medline].
2.	Levine MM, Galen J, Barry E, Noriega F, Chatfield S, Sztein M, et al. Attenuated salmonella as live oral vaccines against typhoid fever and as live vectors. J Biotechnol 1996; 44: 193-196[Medline].
3.	Ferrari G, Humphrey W, McElrath MJ, Excler JL, Duliege AM, Clements ML, et al. Clade B-based HIV-1 vaccines elicit cross-clade cytotoxic T lymphocyte reactivities in uninfected volunteers. Proc Natl Acad Sci USA 1997; 94: 1396-1401[Abstract/Free Full Text].
4.	Polo JM, Belli BA, Driver DA, Frolov I, Sherrill S, Hariharan MJ, et al. Stable alphavirus packaging cell lines for Sindbis virus and Semliki Forest virus-derived vectors. Proc Natl Acad Sci USA 1999; 96: 4598-4603[Abstract/Free Full Text].
5.	Donnelly JJ, Ulmer JB, Shiver JW, Liu MA. DNA vaccines. Annu Rev Immunol 1997; 15: 617-648[Medline].
6.	Belshe RB, Mendelman PM, Treanor J, King J, Gruber WC, Piedra P, et al. The efficacy of live attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine in children. N Engl J Med 1998; 338: 1405-1412[Abstract/Free Full Text].
7.	Nichol KL, Mendelman PM, Mallon KP, Jackson LA, Gorse GJ, Belshe RB, et al. Effectiveness of live, attenuated intranasal influenza virus vaccine in healthy, working adults: a randomized controlled trial. JAMA 1999; 282: 137-144[Abstract/Free Full Text].
8.	Giuliani MM, Del Giudice G, Giannelli V, Dougan G, Douce G, Rappuoli R, et al. Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-labile enterotoxin with partial knockout of ADP-ribosyltransferase activity. J Exp Med 1998; 187: 1123-1132[Abstract/Free Full Text].
9.	Douce G, Turcotte C, Cropley I, et al. Mutants of Escherichia coli heat-labile toxin lacking ADP-ribosyltransferase activity act as nontoxic, mucosal adjuvants. Proc Natl Acad Sci USA 1995; 92: 1644-1648[Abstract/Free Full Text].
10.	Kotloff KL, Noriega F, Losonsky GA, et al. Safety, immunogenicity, and transmissibility in humans of CVD 1203, a live oral Shigella flexneri 2a vaccine candidate attenuated by deletions in aroA and virG. Infect Immun 1996; 64: 4542-4548[Abstract].
11.	Tacket CO, Kelly SM, Schodel F, et al. Safety and immunogenicity in humans of an attenuated Salmonella typhi vaccine vector strain expressing plasmid-encoded hepatitis B antigens stabilized by the Asd-balanced lethal vector system. Infect Immun 1997; 65: 3381-3385[Abstract]. (Correction in Infect Immun 1997;65:3981)
12.	Sizemore DR, Branstrom AA, Sadoff JC. Attenuated bacteria as a DNA delivery vehicle for DNA-mediated immunization. Vaccine 1997; 15: 804-807[Medline].
13.	Hone DM, Wu S, Powell RJ, et al. Optimization of live oral Salmonella-HIV-1 vaccine vectors for the induction of HIV-specific mucosal and systemic immune responses. J Biotechnol 1996; 44: 203-207[Medline].
14.	Mason HS, Ball JM, Shi JJ, Jiang X, Estes MK, Arntzen CJ. Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proc Natl Acad Sci USA 1996; 93: 5335-5340[Abstract/Free Full Text].
15.	Tacket CO, Mason HS, Losonsky G, Clements JD, Levine MM, Arntzen CJ. Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nature Med 1998; 4: 607-609[Medline].
16.	Glenn GM, Rao M, Matyas GR, Alving CR. Skin immunization made possible by cholera toxin. Nature 1998; 391: 851[Medline].
17.	Minutello M, Senatore F, Cecchinelli G, et al. Safety and immunogenicity of an inactivated subunit influenza virus vaccine combined with MF59 adjuvant emulsion in elderly subjects, immunized for three consecutive influenza seasons. Vaccine 1999; 17: 99-104[Medline].
18.	O'Hagan DT. Recent advances in vaccine adjuvants for systemic and mucosal administration. J Pharm Pharmacol 1998; 50: 1-10.