June 1, 2009 (Vol. 29, No. 11)

Goal Is to Move Products through Clinical Trials Quickly and Safely

One year ago, a new technology predicted that significant outbreaks of the H1N1 flu virus would occur within 6 to 12 months. One year later, the H1N1 flu virus triggered a stage 5 pandemic alert from the World Health Organization.

While the ability to accurately predict influenza outbreaks and their locations is extraordinary, the same technology can be used to develop flu vaccines.

The predictions, made by Replikins, were based on correlations of flu virus specimen and Pub Med documentation of major outbreaks during the past 90 years, focusing on concentrations of, and spacings between, replikins—the lysine and histidine residues in the hemagglutinin unit genetic sequences of the eight major genes in the influenza virus.

Sam Bogoch, M.D., Ph.D., chairman of Replikins, says those replikins are strain-specific and determine the virus’ rate of replication. High counts (e.g., seven replikins per 100 amino acids) indicate an outbreak is imminent. Lower counts (four per 100 amino acids) indicate the virus will remain dormant indefinitely. The analysis showed a point-for-point correlation between high replikin counts and outbreaks.

Dr. Bogoch notes that the company’s PanFlu™, a vaccine that interferes with replikins, is ready for clinical trials. A similar vaccine has proved effective in blocking H5N1 transmission among chickens, and has conferred 91% protection against the lethal Taura syndrome in shrimp populations, he points out, adding that because the replikins are conserved, this strategy has the potential to become a broad-spectrum flu vaccine. That vaccine can be manufactured in seven days, he explains.

Novavax plans to create a virus-like particle-based (VLP) vaccine against the H1N1 strain, according to Rahul Singhvi, Sc.D., president and CEO. “Our recombinant VLP technology obviates the need for a live virus seed for manufacturing,” he says. Therefore, “We can cut the cycle time from strain identification to production of the first vaccine to just 10 to 12 weeks.”

The VLPs contain the proteins that make the virus’ outer shell and the surface proteins, without the RNA required for replication. “There’s no genetic material in these VLPs, and they are unable to replicate,” Dr. Singhvi emphasizes.

The vaccine includes three immunologically important proteins (hemagglutinin, neuraminidase, and matrix1) and is matched to the wild-type virus that causes influenza in humans.

Robust Response

In tests against the Indonesian strain of H5N1 flu, “there was a robust immunogenic response,” Dr. Singhvi says. Phase II results shows that 64% of patients had a hemagglutinin inhibition titer above 1:40 at a 90 mcg dose, and 94% of patients showed a neutralizing antibody response against the H5N1 virus above 1:20 at a 90 mcg dose. Importantly, in preclinical studies, a VLP vaccine candidate against the 1918 H1N1 flu virus conferred protection against that strain and, when administered intranasally, also protected animals against a highly pathogenic strain of the H5N1 influenza.

Phase III trials of the seasonal flu vaccine were planned for 2010, but Novavax stands ready to accelerate trials to combat pandemics, Dr. Singhvi says.

Medicago received the genetic sequence for the H1N1 influenza virus strain in late April to begin testing using its virus-like particle technology. This August, it plans to begin a Phase I trial using its technology to target the H5N1 influenza virus. The goal is to prove the safety of a vaccine produced using Medicago’s plant-based technology.

The company relies on a transient expression system to produce recombinant vaccine antigens in the Nicotiana benthamiana plant, according to Frederic Ors, vp, business development. It has the potential to deliver a vaccine for testing one month after the genetic materials from a virus strain are identified and sequenced, he says.

“We immerse the leaves of the plant into a liquid containing the gene sequence in agrobacterium and apply a vacuum for one minute. This removes the air between the cells. The leaf acts like a sponge, drawing in the agrobacterium. The cells produce the protein for four to six days before leaves are harvested,” explains Ors.

Medicago’s VLP technology has the potential to produce pandemic vaccines before a pandemic strikes and will be in a position to supply large volumes of vaccines to the global market once human trials are completed, claims Ors.

In preclinical trials, the vaccine conferred cross protection against several strains of the H5N1 influenza virus, including those from Indonesia, Vietnam, China, and Turkey. “We also are developing a seasonal vaccine to follow our H5N1 vaccine candidate,” Ors says.

Flagellin-Based Approach

VaxInnate is developing an H1N1 flagellin-based vaccine for swine flu. The flagellin stimulates the immune cells in the body to release cytokine and immune-mediator molecules that initiate an immune response, notes David Taylor, M.D., chief medical officer.

VaxInnate uses HA and M2 proteins to stimulate an immune response to influenza virus. HA mutates rapidly, but the M2ectodomain (M2e) has remained virtually unchanged during the past century and across influenza A strains. “The body doesn’t recognize this as immunogenic,” Dr. Taylor says, until it is fused to flagellin.

To induce an immune response, VaxInnate fuses four copies of M2e to flagellin. “Unfortunately, this is the first M2e vaccine and we don’t know how protective it is in humans,” he says. Therefore, the company is determining its protective properties for influenza A. Pre-trials were expected to begin this spring, with a larger, 2,400 patient controlled trial scheduled to start in the autumn.

M2e and HA each can be produced in bacterial expression systems, shortening development time. Dosage, Dr. Taylor says, is one-tenth that required from egg-based vaccines, so “we can make tens of thousands of doses per liter of bacteria.” And, for a seasonal vaccine, he suggests VaxInnate can include four viral strains rather than the usual three.

A scientist at VaxInnate performs an assay to detect the presence of protective antibodies at the company’s New Jersey labs. The firm is working on an H1N1 flagellin-based vaccine for swine flu.

Nanoemulsion-Antigen Mixture

NanoBio’s NanoStat™ vaccine platform mixes an antigen from an influenza strain with a proprietary nanoemulsion, according to David Peralta, vp, COO, and CFO. When administered in the nose, Mary R. Flack M.D., vp of clinical research explains, the vaccine “coats the mucosa” and then is taken up by dendritic cells that deliver the antigen to the lymph nodes, thymus, and spleen, triggering an immune response.

“The dendritic cells are specific for wanting to engulf lipids, so they take up the nanoemulsion-antigen mixture more efficiently than other vaccines,” says Dr. Flack.

In one day, 100 million doses of the emulsion can be made, claims Dr. Flack. “It’s very stable,” Peralta says, and has remained viable for more than three years. Once the nanoemulsion is made, it can be stored so different antigens can be mixed into the nanoemulsion as needed, making it easier to respond to emerging needs.

Phase I clinical trials of the nasal vaccine, NB–1008, are under way. In animal studies, it triggered a robust mucosal, systemic, and cellular immunity without inflammation or safety concerns. In ferrets, the responses were triggered with only 2% of the standard dosage. There are indications that cross protection may ensue.

The vaccine also is being investigated for hepatitis B, Haemophilus influenzae type B, RSV, cancer, anthrax, smallpox, and other diseases. Using the same technology platform, NanoBio is also developing a nasal spray that kills on contact any strain of influenza by attacking the lipid envelope that surrounds the virus, says Dr. Flack.

NanoBio’s short- and long-term approaches to influenza prevention.

Minimizing Adjuvant

Vaxart is developing oral vaccines for seasonal and pandemic influenzas based upon a nonreplicating adenovirus 5 vector that expresses both a target antigen such as HA and a piece of double-stranded RNA that acts as an adjuvant via the toll-like receptor 3. According to Mark Backer, Ph.D., CEO, “Presenting the adjuvant and antigen together to the immune system at the cellular level minimizes the amount of adjuvant needed to get a strong immune response to the target antigen. Unlike most vector approaches, performance is unaffected by previous exposure.”

In preclinical trials, the avian flu vaccine showed a strong immunologic response, even against mismatched strains, suggesting it may remain effective as viruses mutate. Animals were protected against lethal challenge by the H5N1 avian flu virus.

To combat the current H1N1 flu strain, a synthesized HA gene with the matching sequence is being used to produce a vaccine without growing the flu virus itself, letting Vaxart produce a vaccine about two months faster than companies that must grow the virus, according to Dr. Backer, who notes that the production rate is expected to be 20 times higher than flu grown in cell culture.

Because the vaccine is oral, it could be delivered by the postal service and administered by patients themselves. As the work has not yet entered clinical trials, Dr. Backer says it may be most beneficial in the event of subsequent waves of the H1N1 flu.

Pulmatrix has developed a “one drug, many bugs” concept that doubles as a preventive and treatment. “It’s not a vaccine replacement. It’s complementary,” emphasizes Bob Connelly, CEO. He says it’s designed for high-risk situations, populations susceptible to respiratory infections, and for those who already have a respiratory infection from any airborne pathogen, including viral and bacterial sources. “It’s pathogen-agnostic,” he says.

The lead drug candidate, PUR003, is an inhaled cationic airway lining modulator composed of positively charged molecules that are simple and safe. “The formulation triggers both physical and biological effects in the airway,” Connelly says.

Phase II trials for influenza are scheduled for this year, with the first beginning in July.

The enteric coating on Vaxart’s oral vaccine carries the nonreplicating Ad5 vector through the stomach, then dissolves.

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