ILLUSTRATION: TONY FERNANDEZ

Modern vaccine research is at the forefront of human health. Vaccine prophylaxis is the first defense for many incurable and debilitating diseases, from tetanus to typhoid, from polio to plague. For other diseases, it can prove a therapeutic weapon, sometimes the only one remaining (or possible) in a depleted arsenal.

Today, vaccine researchers have a broader vision than ever before. The development of new and better approaches to vaccines is seen not only as the great leveler in the war against infectious diseases such as AIDS and malaria, but potentially the most potent weapon against a host of noninfectious diseases, including asthma, Alzheimer’s disease, and even cancer.

But vaccines are not magic. Like any other drug products, they must be researched and developed for proper use. Traditionally, there have been only a few types of vaccines, made primarily from attenuated or killed pathogens or from attenuated or deactivated toxins, used as antigens to trigger an adaptive immune response. Their effectiveness was discovered mainly by trial and error, for they were born in an era when knowledge of the immune system did not even include the concept of antibodies. Today, through a host of physiological breakthroughs, the new molecular biology, and the tools of genomics, the concept of vaccination has grown to include the induction and modulation of many stages and participants in the immune process.

Enhancing immunity
To determine how and when to modulate the immune response, it is first necessary to understand it. If new or better “pressure points” can be found and exploited in the adaptive immune system, then more effective vaccines can be developed.

All these steps can be considered potential pressure points for controlling and improving vaccine effectiveness. Researchers have tried to increase dendrite production and activation and have used a wide variety of methods of enhancing antigen recognition, uptake, and presentation. Identification and analysis of the ever-increasing number of immune-related cytokines are particularly active areas of research. And finally, various synthetic chemical adjuvants are being investigated for their abilities to influence the process (see box, “Assisting antigens”).

Engineering answers
In the past, most vaccines used attenuated or deactivated versions of the toxin or pathogen that caused the original disease. The possibility of side effects, or even of contracting the disease through bad manufacturing practices, was always of concern. In the modern, far more risk-averse era, any means of reducing the threat of bad outcomes is of paramount interest to vaccine researchers and producers. One of the best ways to prevent the possibility of living contaminants or improperly inactivated enzymes or toxins is to take a genetic engineering approach, by making recombinant vaccines that never saw a pathogen or by using partial gene sequences that cannot produce an active product, only an antigenic one.

The attempt to find an AIDS prophylactic is one of the best examples of using the new genetic engineering technologies for vaccine development. Because the disease is so deadly and so frightening, few researchers seriously consider the use of attenuated live or killed virus as a vaccine, for even if it could be made effective, it probably would not be acceptable to the public. The alternative is to use fragments of the virus—specifically, viral envelope proteins. And the safest way to use such proteins is not to purify them from viruses but to synthesize them separately in bacteria through genetic engineering techniques. AIDS vaccine research is so prevalent that a conference was dedicated to the subject in Philadelphia in September 2001. Research was presented there on a wide variety of vaccine avenues, including almost every possible target site in the virus and the immune system (http://63.84.172.40/).

Among the most promising developments in current clinical trials reported at the conference was the recombinant canarypox DNA (ALVAC 1452, Aventis Pasteur) vaccine, which codes for key HIV proteins, including parts of the envelope known as pol and nef proteins. When used in HIV-positive patients who had discontinued highly active antiretroviral therapy (HAART), the vaccine helped limit viral load rebound. This vaccine could be critical for new avenues of treatment in which HAART is discontinued to help eliminate drug-resistant HIV strains from a patient’s system.

But infectious diseases are not the only promising road for vaccination. Developmental or genetic diseases, such as Alzheimer’s, also may be amenable to a vaccine-based approach. Researchers have shown that mice genetically engineered with the human gene to develop the disease could be protected by a vaccine consisting of an engineered peptide fragment of the amyloid plaque protein. Similar vaccines are being tested in early human clinical trials (1).

DNA and Darwin
There is currently considerable excitement in the field of DNA vaccines. Unlike the live or attenuated pathogens and their toxins or protein components, which are traditionally used in vaccines, DNA vaccines employ the naked DNA that codes for antigens rather than the antigen itself. Injections of plasmid DNA have proved capable of stimulating both the humoral and the cellular immune systems. In animals, DNA vaccines have provided effective immunity against a wide variety of viruses, bacteria, and parasites (2). Recently, DNA vaccines have become an exciting area of research in the development of cancer vaccines.

In a timely note, researchers at Ohio State University reported in October 2001 that they had successfully immunized mice against anthrax infection by using a plasmid DNA vaccine containing the coding region for either or both of two proteins critical to forming the anthrax toxin. Mice were tested with 5 times the lethal dose of the toxin. All the mice that had received the plasmid injections were immune, whereas the control mice died within several hours (www.osu.edu/researchnews/archive/anthrax.htm).

DNA vaccines are also being used in the attempt to conquer AIDS. In October 2001, the National Institute of Allergy and Infectious Diseases began Phase I clinical trials of its first vaccine, which contains DNA for the gag and pol genes. Gag is HIV’s core protein, and pol includes three enzymes crucial for HIV replication. All the DNA sequences were modified to render the vaccine safe. Gag and pol are considered good candidates for developing AIDS vaccines because they are relatively constant across different virus strains and account for a large percentage of total virus protein (www.niaid.nih.gov/newsroom/releases/vaccinemade.htm).

Recently, scientists have even begun to combine Darwinian selection with the DNA approach in an attempt to develop optimal vaccines. The selection process is known as directed molecular evolution. In one example, presented at the 11th International Congress of Immunology in July 2001, Juha Punonnen, vaccine director at Maxygen (Redwood City, CA), outlined the company’s attempts to create a vaccine for Dengue fever (none is currently available).

Mosquitoes transmit the virus that causes Dengue fever and widespread destruction in Africa. The virus exists in four distinct antigenic strains, so that a vaccine against only one strain would be ineffective against the others. Using a technique called DNA shuffling, the researchers generated chimeric antigens for vaccine testing by splicing and screening DNA from the envelope genes of the four different strains of Dengue. DNA from each of the four envelope protein types was fragmented, and a PCR-like reaction was used to reassemble the fragments. Mice were injected with the novel DNA sequences produced, and a few of the clones tested induced an antibody response to all four antigens. Whether the new DNA vaccine protects the mice against Dengue fever has yet to be determined, but many consider the approach promising. DNA shuffling techniques are also being used to investigate the production of new cytokines in an effort to further control and modulate cellular responses, including the immune response. A review of DNA shuffling and its role in vaccines is available (3).

Dendrites and Co.
Manipulation of the cell-based immune system through modulation of the T-cell system, as well as of dendrite production and behavior, is also a promising area of vaccine research. This is especially true in the realm of cancer vaccines. In a recent example, the immune-stimulating hormones granulocyte macrophage-colony stimulating factor (GM-CF) and interleukin-4, which trigger the transformation of blood monocytes into dendritic cells, have proved useful in in vitro studies. At the University of California, Los Angeles, Johnson Cancer Center, these hormones have been injected into cancer patients, in whom they stimulated dendrite formation. This procedure may be adapted for use as therapy someday. Chimeric fusion proteins containing GM-CF have shown T-cell-mediated antitumor effects against renal carcinoma (4). In other cases, dendrites have been vaccinated outside the body with cancer antigens, stimulated to grow to large numbers in vitro, and then reinjected to provide a significant boost to the immune system’s attack on a variety of cancers, from brain tumors to melanomas.

Envisioning the future
Ultimately, the key promise of vaccines lies in the application of a protective system that nature has honed over many hundreds of thousands of years of animal evolution. From the use of dendritic cells to the development of new adjuvants to the use of recombinant DNA vaccines, modern molecular biology has learned to manipulate the immune system already extant in human beings to therapeutic and prophylactic ends. With the addition of autoimmune diseases, inherited disorders, and cancers to the mix, the use of vaccines promises to be one of the most profitable ventures (in both human health and dollar value) in the pharmaceutical industry. These time-honored mementos of the 19th century and Pasteur’s greatness, updated and transformed, may turn out to be some of the most valuable contributions of 21st century medicine to the human condition.

References

1. Sigurdsson, E. M.; et al. Am. J. Pathol. 2001, 159, 439–447.
2. Lowrie, D. B.; Whalen, R. G. DNA Vaccines: Methods and Protocols; Humana Press: Totowa, NJ, 2000.
3. Whalen, R. G.; et al. Curr. Opin. Mol. Therap. 2001, 3, 31–36.
4. Tso, C.-L.; et al. Cancer Res. 2001, 61, 7925–7933.

Mark S. Lesney is a senior editor of Modern Drug Discovery. Send your comments or questions regarding this article to mdd@acs.org or the Editorial Office by fax at 202-776-8166 or by post at 1155 16th Street, NW; Washington, DC 20036.