Publications Archive

A Boosted Hope: AIDS Vaccine Research

by Marjan Hezareh, Ph.D., ARA's Director of Scientific Communications

The human immunodeficiency virus (HIV) continues to spread around the world, insinuating itself into communities previously little troubled by the epidemic. Already almost 20 million people have died of AIDS, over 30 million are currently living with HIV and 16,000 new infections occur daily. HIV chemotherapy has continued to allow substantial improvements in quality of life and life expectancy for HIV infected individuals. However, these therapies are out of reach for most HIV-infected patients around the world. Even when drugs are available they are limited by their toxicity, complex regimens and elevated cost. Therefore, the biomedical community is redirecting efforts towards development of an effective AIDS vaccine. In this issue of Searchlight, we review some of the challenges facing the scientific community in designing and developing a safe and effective vaccine against HIV.

The term immunity is derived from the Latin word immunitas referring to the exemption from variety of civil duties offered to Roman senators during their tenures in office. Historically, immunity meant protection from disease—specifically infectious disease.The modern definition of immunity is a reaction to foreign substances, including microbes, viruses, and macromolecules such as proteins and polysaccharides. The concept of immunity existed long before, as suggested by the ancient Chinese custom of making children inhale powders made from the crusts of skin lesions of patients recovering from smallpox. Edward Jenner's successful vaccination against smallpox demonstrated for the first time that the immune system can be manipulated under controlled conditions. He noticed that milkmaids who had recovered from cowpox never contracted smallpox. He established that cowpox virus cross-reacts immunologically with smallpox virus and could be used to protect against smallpox. This led to the widespread acceptance of vaccination for inducing immunity to infectious disease.

Unfortunately, there is no population that has been cured from AIDS. Therefore, no immune parameters have been identified that correlate with protection from disease. Recent studies on exposed seronegative sex workers in Nairobi provided important insights into the mechanisms the body uses to maintain immunity, but it failed to identify definitive markers to guide vaccine development. Although immune correlates for protection against HIV remain unknown, there is evidence that both cell-mediated immunity and long term B-cell memory are crucial in immune protection. Furthermore, transmission of HIV occurs through multiple routes. Therefore, an effective vaccine may require stimulation of all major immune response mechanisms including humoral (antibody response), cellular (cytotoxic T cells and helper T cell), and local (mucosal response at the site of the infection). Other vaccines such as the childhood vaccine for the prevention of pertussis have been successfully developed without precise knowledge of correlates of immunity. Development of a vaccine without a definitive marker of protection is possible, but remains one of the more challenging aspects in AIDS vaccine development.

The genetic diversity of HIV poses another difficulty. HIV has multiple sub-types or clades, around the world, and even in an individual mutations occurs very quickly and with a high frequency. Efforts to develop a universally-effective vaccine against all strains and clades of virus have been impeded by the unusual diversity of the HIV virus. Other vaccines have been designed that successfully protect humans against pathogens where strain variation occurs, such as pneumococcus, influenza, and polio. But the strain variation of these viruses is relatively limited compared to HIV.

Further barriers to vaccine development include the fact that there is no ideal animal model for HIV/AIDS. Vaccine studies for SIV on monkeys have provided important insights into HIV immunopathogenesis, but there are differences between HIV and SIV in their genomic organization that could affect vaccine efficacy. Although the animal models are not perfect they help determine the mechanisms of protection and may help us to make decisions about proceeding with human trials. Furthermore, we should keep in mind that other vaccines, including measles, mumps, and rubella, have been successfully developed where animal models were not available or less than ideal. The true test of a vaccine candidate will be determined in large human clinical trials, not in animal models.

Recent advances in immunology have led to the design and development of new and promising vaccine strategies. As summarized below, many of these strategies are being tried to develop an effective HIV vaccine. Some scientists are calling for a return to traditional methods of vaccine development such as live attenuated vaccine, which have greater risk but may have a greater chance of success. Others are pushing for the use of high-tech methods because of the unique features of HIV. Either way, human clinical trials are the crucial link between laboratory research and an effective vaccine. It takes lots of determination and a series of vaccines and human clinical trials to develop a product capable of affecting the epidemic. Although the hurdles to development of an effective HIV vaccine are high, we should be heartened by the renewed interest in vaccine development.

Current AIDS Vaccine Candidates

Several types of vaccines are currently in development, either in human clinical trials or in experiments with primates.The following list summarizes different concepts currently in uses for development of an AIDS vaccine.

Recombinant Subunit Vaccines: In this strategy, individual viral components are used to generate a protective immune response. This is the basis of AIDSVAX™ B/B, the first vaccine currently in Phase III clinical trials. This vaccine contains synthetic copies of gp120 (HIV envelope) protein.

DNA Vaccines (or "naked DNA" or "nucleic acid"): contains an HIV immunogen under the control of an eukaryotic enhancer/promoter signal that confers an appropriate expression of the viral protein. Once introduced into muscle, cell surrounding the site of injection, internalize the plasmid and transport the DNA into nucleus, where appropriate expression of immunogene will occur. This is the basis of Merck's "naked DNA" vaccine currently being tested in Phase I clinical trial.

Viral or Bacterial Vector-Based Vaccines: Recombinant vector-based vaccines contain replication-defective forms of non-pathogenic virus (e.g. Poxvirus, Modified Vaccinia Ankara) or bacteria (e.g. Salmonella thyphosa) to carry one or more HIV genes. This is the basis of Aventis's vaccine candidate, in which a harmless canary poxvirus has been used as a vector.

Live-Attenuated Vaccines: This concept is used to protect humans against broad range of infectious disease such as polio and measle. Monkeys infected with live-attenuated SIV (nef-deleted Simian Immunodeficiency Virus) were not infected upon subsequent challenge with SIV or nef-deleted SIV. However, it was found that significant pathology occurred in monkeys after exposure to live-attenuated SIV. Furthermore, nef-mutated virus has been isolated in some patients infected with HIV. This approach may prove to be effective, but many safety concerns should be addressed before testing such a vaccine in human clinical trials.

Whole-Inactivated Virus: This concept is used to protect humans against broad range of infectious disease such as polio, influenza, and rabies. In this approach virus is inactivated using different methods including chemicals, irradiation or heat. Inactivation of virus insures that every virus particle is killed while they maintain their primary structure necessary to stimulate HIV-specific immune response. A vaccine candidate is being developed at UCLA by Dr. Katie Grovit-Ferbas and at NCI by Dr. Larry Arthur based on killed, whole-inactivated HIV virus.

Virus-Like Particle Vaccines: These vaccines are made of pseudovirion that contain non-infectious portions of HIV viral particle but lack most or all of HIV genetic material. The advantage of this approach lies on the fact that HIV proteins are presented in their natural forms to the immune system. However, it is very difficult to prove that no infectious particles exist in the vaccine preparation.

Peptide Vaccines: Consist of chemically-synthesized pieces of HIV protein (peptide) known to stimulate HIV-specific immunity.

AIDS Vaccines in the New Millennium: Meetings Updates

By Marjan Hezareh, Ph.D.

Researchers at both the "AIDS Vaccines in the new Millennium" conference in Keystone (28 March-3 April) and at the "AIDS Vaccine 2000" in Philadelphia (September 5-8) presented a broad range of studies on HIV vaccines candidates. The need for a vaccine has never been greater and important progress toward this goal was presented during these meetings. NIH has increased HIV vaccine research funding more than six-fold since 1990, to an estimated $356.6 million for fiscal year 2002. At NIAID (NIH institute for HIV vaccine research), an estimated $450.7 million will be devoted to all vaccine research in year 2002, with 61 percent of that total ($276.5 million) dedicated to HIV vaccine development.

"The field of vaccine research owes great thanks to the largesse of the American people, the Administration and Congress for making these resource available," says Dr. Fauci in his keynote lecture in Philadelphia. The data being discussed demonstrated an increasingly complex collection of vaccine candidates and approaches in development, which offer a new basis for new optimism. A better knowledge of envelope structure allowed researchers: 1. to define new HIV antigen structures to induce useful immune response and 2. to better understand how the immune system first recognizes and responds to HIV.

All together, these advances helped to design new strategies that may provoke efficient antigen uptake and presentation leading to better T-cell activation. A growing number of studies were directed toward the role of mucosal immunity in HIV protection and the probability of including mucosal immunity as a component of vaccine-induced protection. Like the skin, the mucosal epithelia are barriers between the internal and external environments, and are therefore an important first line of defense, specifically in HIV infection that is transmitted sexually. Furthermore, the ongoing phase I and II trials, and diverse vaccine approaches including gp120 and canarypox vectored HIV-1 candidate vaccines (ALVAC) provided increasing clarity about the safety and immunogenecity thresholds required to take a vaccine from animal studies into human trials and Phase III evaluation of efficacy.

Here we summarize some important data on HIV vaccine development presented at these conferences.

Protection with Adeno-Associated Virus Vectors

Philip Johnson from Children's Research Institute, Columbus, Ohio, showed data from ongoing studies of an SIV vaccine based on vectors of recombinant Adeno-Associated Virus (rAAV). Wild type AAV is a replication defective parvovirus, which is non-pathogenic in humans and animals. In this study, they generated rAAV vectors (which lack any AAV gene) expressing SIV env, rev, gag and protease gene and used them to immunize intramuscularly 8 monkeys (single dose). They detected a strong CTL response and persistent neutralizing antibody titers over 14 months. After immunization, animals were challenged intravenously with a high dose of SIV-E660. In vaccinated animals, viral peak was reduced by about 1.3 log and viral load by 3 logs. After 6 months, vaccinated animals were still healthy, while 3 out of 8 control animals died. Based on these data, rAAv vectors stimulate robust, durable and effective immune responses against SIV. In addition, they demonstrated that immunization with DNA prime/rAAV boost stimulate better immune responses that rAAV alone. Dr. Johnson suggested that these data provide evidence necessary to move forward the clinical trial of cognate rAAV/HIV vaccine in humans.

Protective immunity induced by whole recombinant Yeast-Based HIV Vaccine

Dr. Franzusoff from GlobeImmune, Inc., Denver, presented data on the development of a novel therapeutic vaccine based on using, whole, recombinant, non-pathogenic yeast (Saccharomyces Cerevisiae) as a vector. The optimal stimulation of CTLs requires presentation of antigens by Dentritic Cells (DCs). These cells are unique in their ability to process antigens into the MHC class I pathway for presentation to CTLs. In this study they engineered recombinant yeast expressing HIV-1HXB2 p55 gag protein (HIVAX-2). mice (C57B1/6) were immunized subcutaneously with 20 million intact live or heat-killed yeast.

The vaccination stimulated strong immune response in mice, measured by intracellular IFNg staining, by CTL-mediated cell lysis and by T cell proliferation assays. To show protective immunity, they used HIV-gag dependent tumor model. Mice were protected against subsequent challenge with B16 melanoma cells stably expressing HIV-1HXB2 p55 gag. Therefore, the recombinant yeast-based HIV vaccine is a potent activator of cellular-immune responses and is currently being prepared by GlobeImmune, Inc. for use as a therapeutic HIV vaccine in a phase I clinical trial.

Merck's DNA and Adenovirus-Based Vaccine

Merck researchers have developed two types of vaccine; one based on "naked DNA" and the other on an adenovirus vector. At the Keystone meeting they presented pre-clinical studies that led them to move these 2 vaccines candidate into phase I trials. In his presentation, John Shiver, Director of Vaccine Research showed data from comparative studies of several HIV vaccine approaches. All vaccines contained SIVgag gene but no env gene. In this study rhesus macaques were immunized with one of the following vaccines: plasmid DNA in saline, alum or adjuvant (CRL10050 adenovirus type 5 (Ad5)-based viral vectors Modified Vaccinia Ankara (MVA)-based viral vector. Each vaccines were administrated intramuscularly to 3 animals 3 or 4 times over 32 weeks. Immunization with the Ad5 based vector resulted in the highest immune response. Response to plasmid DNA in saline or alum was much lower than DNA in adjuvant (CRL1005), showing a clear advantage of adjuvanting DNA. CD4 responses were induced by all vaccines and were higher in the DNA group. The Ad5-based vaccine tends to induce higher CD8 responses. Three months after immunization animals were challenged with a high dose of SHIV89.6P, a very pathogenic lethal strain. At day 180 after challenge all vaccinated animals were alive (although infected), while 4 out of 6-control animals had died. In Ad5 groups, CD4 count remained high and viral load slowly brought under control and peak viremia was reduced by 1.5 log. This group showed the best clinical course followed by adjuvanted DNA with CRL1005 and MVA-based vaccine. Vaccination with DNA in saline or alum resulted in a strong drop in CD4 cells count and the poor control of viremia.

Next, Emilio Emini, reported on pre-clinical evaluation of Merck's HIV vaccine candidates currently in clinical trials. In his presentation he addressed the issue of pre-existing immunity to adenovirus which could make this a less immunogenic vector. About 10% of the US population have significant neutralizing antibodies to adenovirus and 40% of them shows lower level of antibody. Therefore they preexposed 6 monkeys to adenovirus and then immunized them with a high dose (1011 particles) of Ad5-gag (HIVgag). The presence of neutralizing antibodies reduced by several folds the T-cell responses but did not eliminate it. He also presented data on a DNA prime/Ad5 boost study. In this study animals were given first DNA/CRL1005 adjuvant and then boosted with a high dose (107 particles) of Ad5-gag (HIV gag). The vaccinated animals showed a 5-10 fold increase in the number of HIV-specific-T cells response after the boost. This is generally reflected by an increase in CD4 and CD8 T cell response, again CD8 response predominating after Ad5 boost. Thus the preexisting immunity could be overcome, especially if using DNA prime/Ad5 boost strategy. Vaccination with DNA in saline or alum was a less effective prime showing little T cell response.

Emini suggested that a 109 particle dose should still induce a good T cell response even if neutralized by 99%. He also said that nef and pol would be added to Merck's vaccines in addition to gag. This is based on data showing that HIV infected people with good immune responses to these proteins maintain lower viral load. A DNA gag trial began last year in uninfected people and a trial in HIV-infected people started in March 2001.

Importance of mucosal CTL to prevent mucosally-transmitted SIV/HIV

In his study, Jay Berzofsky from the National Cancer Institute, Bethesda, compared mucosal versus systemic immunization in macaques. They demonstrated that mucosal, but not systemic immunization, protected macaques against an SIV challenge. In this study animals were immunized intrarectally (ir), or subcutaneously (s.c.) with peptide HIV vaccine including SIV gag and pol epitopes. After immunization, animals were challenged with SHIV-ku and monitored for 200 days. All animals showed similar viral peaks after challenge. However, following this peak, the viral loads dropped to undetectable levels in ir-immunized animals and they maintained a high CD4 count up to 200 days post-infection. In contrast the sc-immunized animals had high viral loads and a low CD4 cell-counts. All immunized animals showed some degree of protection compared with control animals. Two hundred days post-infection, animals were sacrificed and HIV level in colon and jejunum was monitored. Little or no virus was seen in the gut tissue of ir-immunized macaques, while 10-100 times more virus has been found in control and sc-immunized animals. The ir-immunized animals displayed a higher level of HIV-specific CTLs in their colon than sc-immunized animals.

These data is in agreement with Berzofsky's study in mice. In this study they showed a clear relationship between mucosal vaccination, high level of mucosal CTLs and protection against subsequent mucosal challenge. Berzofsky's study is the first showing a correlation between mucosal immunization and improved protection in primates. Future Directions Why there are relatively few studies on mucosal immunity? This is mainly because analysis of mucosal immune activity requires an invasive procedure, while systemic immune activities can be easily measured from blood samples. Several groups are developing techniques to use homing markers as a way of measuring mucosal response in blood. Homing markers are cell surface molecules that indicates destinations of cells in blood stream. Therefore, it is possible to monitor cells bearing these markers from blood samples rather than from mucosa.

One example of such a marker is alpha4beta7, the latter is an integrin that appears on all cells trafficking to the gut. More research has been focused on understanding the early events in HIV infection, such as the role of dentritic cells in the mucosa. It appear that these cells carry HIV to lymph nodes and from there virus spread to other sites. More effort has been focused on understanding the mechanism of action of DC-SIGN, the receptor that carries HIV to lymph nodes, and to find a way to inhibit its action. Although a vaccine that prevents or delays HIV infection remains an elusive goal, more good news is coming our way.

"Perseverance is a great element of success. If you only knock long enough and loud enough at the gate, you are sure to wake up somebody."

—Henry Longfellow