HIV Drugs Produced from Goats Show Promise

Molecular mimicry is a common, but undesirable, property of disease proteins. Sequence similarities between cancer proteins and self-proteins allow tumor cells to hide, thereby avoiding attack from the immune system. HIV uses the same evasive tactic.

In the early 1990s, Dr. Frank Gelder (then director of the Histocompatibility Testing Laboratories, Department of Surgery, at Louisiana State University Medical Center) was researching ways to develop neutralizing antibodies. He reasoned that a therapeutic derived from another species might circumvent one of HIV's major defensive strategies - disguise.

Gelder's inspiration to go across species stemmed from a 1992 clinical trial using human antibodies. Researchers had planned to infuse plasma with high levels of HIV-neutralizing antibodies from a small group of patients who were HIV-positive but never developed clinical signs of the disease.¹ The approach was largely abandoned when researchers discovered that the virus could mutate into forms resistant to neutralizing antibodies.²

Gelder's hypothesis required testing on several species. Goats emerged as the optimal species.

Goats are well suited to biopharmaceutical production because of their large size and availability. Other advantages include their relatively short gestation period (five months, whereas cows need nine months) and tractability, which is essential because of the extensive handling required of animals used in drug production. The real importance is that goats can produce antibodies to unique HIV epitopes that are invisible to the human immune system.

Gelder eventually learned of Dr. Wayne Watkins, a University of Auckland professor. Watkins was conducting similar research, raising antibodies in goats and other farm animals. In the mid-1990s, Gelder moved to New Zealand, and the two founded Virionyx. The company's first product, the antibody HRG214 (HRG in this article), is intended for treatment of AIDS patients who no longer respond to medication such as highly active antiretroviral therapy (HAART). Currently HRG is in early-stage clinical testing in the United States and Australia.

Establishing the Herd

New Zealand's biopharmaceutical industry is based on farm animals. Several companies maintain herds in or source parent stock from New Zealand. Among these are New Zealand-based PPL Therapeutics in Walkato, AgResearch in Ruakura, and Diatranz in Auckland and US-based Invitrogen in Carlsbad, CA. Hyclone, headquartered in Logan, UT does contract work.

New Zealand's geographical isolation is an advantage for biopharmaceutical development in animals. The country has strict biosafety regulations and a disease-free status for major livestock infections that have devastated herds in other parts of the world. There have never been outbreaks of foot-and-mouth disease, bovine spongiform encephalopathy, rabies, or a host of other diseases. The country has been free of scrapie and anthrax since 1954.³

Animal husbandry for the biopharmaceutical industry is a specialized skill. Virionyx opted to concentrate on processing and contract maintenance of its herds to two other companies. Separate herds are maintained in geographically remote regions for security reasons and to further minimize the risk of disease. One herd is at South Pacific Sera, based in the Southern Alps of the South Island. The second is maintained by HyClone New Zealand on the North Island. These companies will be key partners as HRG moves towards commercialization.

FDA and New Zealand's regulatory agencies enforce rigid standards in animal husbandry, plasma extraction, record keeping, and quality control. Herds must be raised in isolation from other animals. Qualified personnel must monitor everything about them, each day of their lives, including their genetic background, health status, and post-production fate.

All of the husbandry companies' activities are carefully scrutinized by government agencies, including animal ethics committees. Their practices are well understood and humane. The goats are well cared for and none of the procedures are considered too invasive or too far removed from normal farm practice. Consequently, they do not pose any concerns to animal welfare groups. There are no environmental issues because very little waste is generated during routine animal care, immunization, and plasmapheresis.

The Virionyx herds have been developed from a closed herd of Saanen dairy goats and are the product of conventional breeding practices. The kids are taken at birth and hand-reared inside sheds. This precaution helps avoid contact with common diseases, such as caprine arthritis encephalitis virus and Johne's disease (Mycobacterium avium subsp. Paratuberculosis). Goats are most susceptible to these diseases in the first few days of life. Raising the goats by hand also makes them comfortable with being handled. This is critical in animals that will provide blood on a regular schedule.

When the goats are between 16 and 20 weeks old, they are turned out to graze in doubled-fenced paddocks to ensure they are protected from contact with other farm animals. Goats must be at least seven months old and weigh at least 30 kg to qualify for the immunization program. While actively producing plasma they are inspected daily and weighed monthly to ensure there is no loss of condition. The farm must pass regular independent checks by animal ethics veterinarians, and the goats are under the care of a consultant veterinarian at each farm. The animals are fed dietary supplements to ensure there is no anemia or any other problem associated with the regular donation of plasma.

Immunization and Processing

As soon as it enters the program, a goat is immunized with HIV proteins purified from inactivated viruses. The goats receive booster shots on a regular schedule (every two weeks to every two months for a period of five to seven months) with HIV proteins and synthetic peptides. The HIV proteins are derived from selected strains of the virus grown in cultured cell lines; the virus is purified and lysed to yield released-proteins in solution. The synthetic peptides, made using standard procedures, have amino acid sequences that represent regions on HIV and are designed to amplify the immune responses to specific targets on the virus. Some regions of the virus fail to provoke an immune response in humans, but they do provoke an immune response in animals.

After 12 weeks, the goats are bled and their plasma is tested for immune response to HIV. The initial tests are a simple Western blot assay and an enzyme immunoassay to quantify antibody levels. Doctors use these tests to diagnose HIV in human patients; they are slightly modified to work on goat plasma. The Western blot assay is based on Genelabs' HIV Blot 2.2 kit, but the signal-generating second-antibody conjugate supplied in the kit was changed to detect goat antibodies instead of human antibodies. The enzyme immunoassay uses components in the Abbott Diagnostics gO EIA kit, except buffers were modified to decrease nonspecific effects.

Plasma that meets the acceptance criteria for these tests undergoes a cell culture assay to determine its ability to neutralize HIV. This assay predicts how the antibody will function in patients.

This process of boosting and testing immune response continues until the plasma contains high concentrations of antibodies targeted to at least seven or eight HIV epitopes not recognized by the human immune system. Then, plasma is collected through plasmapheresis under strict blood-banking procedures in a fully closed and sterile system. The plasmapheresis system currently used is a Baxter autopheresis machine, which is connected by catheter to the goat's jugular vein. It collects whole blood, separates the cells from the plasma by centrifugal action, and then returns the cells to the goat's circulation along with a small amount of anticoagulant. Plasma is retained in the collection bag, which is sealed, wrapped, and frozen for storage. New catheters, collection bags, and tubing sets are used for each animal.

The plasma bags are frozen and shipped to the facility in Auckland, where they are put through a standard fractionation process. Goat plasma is similar to human plasma, so the fractionation process is basically the same. Caprine IgG is extracted from the pooled plasma using immunoglobulin-specific ligand affinity chromatography, which yields 90 to 95% pure product. After adjustment of pH and salt concentration, it is then purified to >98% IgG by ion exchange chromatography. The purified IgG is formulated by buffer exchange and diluted to the required concentration. The company works in 25-L batches of plasma (requiring about 50 bags) to yield 50 to 60 L of finished product. The projected antibody concentration for each batch is based on bag-by-bag analysis.

The company currently has a scale-up plan in place for a 200-L plasma batch size. A new facility adjacent to the current laboratory will be built to accommodate the enlarged plant. This is expected to take 18 to 24 months, depending on overall progress in the clinical development program and the final phases of the process development plan, particularly validation.

The key challenge Virionyx faces is the herd. Work is underway to quadruple the herd in the next two years, increasing the number of goats from the current 600 to several thousand. The logistics of obtaining, immunizing, and maintaining so many animals are significant but certainly not impossible to overcome. All specifications for the product have been established and a quality-management program is in place.

Toxicity Testing

Our firm, like all companies producing animal-based products, was required to meet stringent safety standards, detailed in several sets of manufacturing guidelines prior to the start of clinical trials in March 2001.

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These guidelines are designed to minimize the risk of cross-species infection and other problems related to nonhuman material. They cover all aspects of the production of biopharmaceuticals from nonhuman sources, from animal husbandry to surveillance for infectious agents (such as culture tests for bacteria, fungi, and mycoplasma and PCR probes for microorganisms).

One of the most critical steps is removing antibodies that could have adverse effects on patients. Some of these antibodies are stimulated in the goats by human proteins that were incorporated into the viral envelope from infected cells during the budding process. The human host cell proteins are copurified into the viral protein immunogen mixture used in the goats. We minimize the host cell proteins by passage of the viral protein mixture through an affinity column containing immobilized antibodies. The immobilized antibodies are crossreactive against the host cell proteins and capture these proteins but not the viral proteins, which are processed into the immunogen.

In addition, antibodies that are crossreactive to human targets occur naturally in goat plasma and can only be removed through affinity absorption. Selected human proteins are immobilized on the affinity matrix, and goat plasma is passed over the matrix to remove the unwanted antibodies. The process does not affect the desired active antibodies, which are not reactive against the immobilized proteins.

HRG is then formulated into an intravenous solution. We specify 10 mg/mL of caprine IgG antibodies in a sterile saline solution with a pH 6.0 to 7.5. Final packaging is completed onsite in 50-mL glass vials. The final product is tested - according to FDA guidelines in an FDA-approved laboratory - for HIV antibody activity, sterility, toxicity to human red and white blood cells, and biological activity. This process takes about four days per batch.

In Vitro and Clinical Data

The medical properties of HRG are of interest. (Because drug is not yet FDA-approved, we must inform readers that all legal disclaimers are implied.) HRG contains caprine antibodies to at least seven HIV epitopes found on the external envelope proteins gp120 and gp41, as well as the core proteins p24, p66, p17, and p11. These epitopes are not recognized by the human immune system.

An early series of compassionate-use trials treated 77 people in the United States, Mexico, and New Zealand. The results suggested that more studies would be appropriate. Because of the uncontrolled nature of the trials it was impossible to draw firm conclusions about the efficacy of the treatment.

In vitro studies conducted in Australia at the Westmead Millennium Institute in Sydney, and the Macfarlane Burnet Institute for Medical Research and Public Health in Melbourne, (both partners of Virionyx) demonstrated that HRG provoked a consistent, reproducible neutralizing effect, reducing levels of p24 by 90%. The antibody neutralized a broad spectrum of HIV-1 strains, and this activity was increased by the addition of human complement, a protein within the blood stream that works with antibodies to kill invaders.⁸ Preclinical toxicology studies in mice and rabbits found no toxicity in animals receiving 24 doses of HRG over a 30-day period.⁸

In 2000, Dr. Bruce Dezube, associate professor of medicine, Harvard Medical School, became interested in HRG because its mechanism of action differs from any compound, either investigational or approved, to fight the infection. He considers HRG to be unique because it works through seven or eight targets, theoretically overcoming HIV's ability to mutate rapidly and develop strains resistant to single target agents (a stumbling block observed in monoclonal antibody therapies). The immunization process, which includes several rounds of priming the goats with peptide boosters, produces an antibody with strong anti-HIV reactivity.

Dezube conducted a phase 1 study at Harvard Medical School's Beth Israel Deaconess Medical Center, in Boston. The study, designed to examine toxicity and pharmacokinetics, enrolled 18 HIV patients with CD4 counts of at least 50 cells/μL and HIV-1 RNA levels greater than 500 copies/mL. Patients received a single injection of 1, 2, 4, 8, or 16 mg/kg of body weight. The most common side effect was transient rash, with seven patients developing grade 1 or 2 rashes and one developing a grade 3 rash; all cleared up on their own or with an over-the-counter antihistamine. A single dose achieved adequate plasma concentrations well above those that produced an inhibitory effect on HIV in the in vitro studies. At the highest dose, the median half-life was over 68 hours. Although the study, completed in March 2002, was not designed to gauge efficacy, the average decrease in HIV was at 0.25 logs of viral load, and some participants registered up to 2.6 logs.⁹ The tests that exceeded 1.0 logs are the impetus for further testing.

Another dosing study was conducted in Sydney by a team from St. Vincent's Hospital, the National Centre in HIV Epidemiology and Clinical Research, and Westmead Millennium Institute. Eleven HIV-1 infected men took part in the phase 1 open-label, nonrandomized, dose-escalation trial. As in the Boston study, a single injection of 4, 8, 16, 32, or 64 mg/kg was well tolerated and achieved concentrations comparable to those that neutralized HIV in vitro.

Both phase 1 studies supported moving on to a six-month multidose treatment program. Dr. Dezube is currently conducting a multiple-dosing trial, which will ultimately enroll 40 patients, to establish the safe, efficacious dose for longer-treatment regimens.

A novel approach to the treatment of AIDS, HRG is a passive immunotherapy based on antibodies isolated from the blood of HIV-immunized goats. This approach provides a stable, relatively inexpensive source of the therapeutic, but it also poses unique challenges in producing a drug that will pass the tests of safety and efficacy in humans. Pending the progress and results of clinical trials, the latter part of 2006 is targeted for release of the drug.

References

1. Vittecoq D, Mattlinger B, Barre-Sinoussi F, Courouce AM, Rouzioux C, Doinel C, et al. Passive immunotherapy in AIDS: A randomized trial of serial human immunodeficiency virus-positive transfusions of plasma rich in p24 antibodies versus transfusions of seronegative plasma.

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2. Poignard P, Klasse PJ, Sattentau QJ. Antibody neutralization of HIV-1. Immunology Today 1996; 17:239-246.

3. Belton D. New Zealand's freedom from specified animal diseases [Memorandum]. Ministry of Agriculture and Forestry Biosecurity Authority 2003 24 Jul; Ref. AS00-100.

4. CDC. US Public Health Service guideline on infectious disease issues in xenotransplantation. MMWR Recomm Rep 2001 Aug 24; 50(RR-15).

5. FDA. Guidance for industry: for the submission of chemistry, manufacturing and controls and establishment description information for human plasma-derived biological products, animal plasma or serum-derived products. Rockville (MD); 1999 Feb.

6. EMEA. Note for guidance on production and quality control of animal immunoglobulins and immunosera for human use. London; 2002 Jul 24.

7. FDA. Biological products: general - physical establishment, equipment, animals and care. Code of Federal Regulations, Title 21, Part 600.11 2002 April 1.

8. Williams LA, Haddad D, Choy V, et al. HRG214: A passive immunotherapeutic with anti-HIV neutralizing activity in vitro. Abstract for Keystone Symposia; 2003; Banff, Canada.

9. Dezube BJ, Proper J, Zhang J, Choy VJ, Weeden W, Morrissey J, et al. A passive immunotherapy, HRG214 in patients infected with human immunodeficiency virus: a phase 1 study. Journal of Infectious Diseases 2003; 187:500-503.