January 1, 2005 (Vol. 25, No. 1)

Focus on HIV, Influenza, Cancer, and Bioterrorism

The flu vaccine shortage plaguing the U.S. this year has drawn considerable media attention, public criticism, and finger-pointing, yet the fact remains that with the 2005 flu season approaching its peak, new flu vaccine producers, novel and cost-effective methods of flu vaccine production, and techniques that will enable rapid, in-season manufacturing of additional doses to meet demand are urgently needed (see “Fluvirin Vaccine Production Issues,” GEN, November 1, p. 1).

Renewed interest and funding for flu vaccine strategies will have to share center stage with ongoing R&D efforts on a variety of vaccine targets including the continuing focus on HIV vaccines, immunotherapeutic-based cancer vaccines, infectious targets with the potential for bioterrorism, and vaccines against common infectious agents such as human papillomavirus (HPV).

A clinical study recently published in The Lancet (364:1757-1765) demonstrated the effectiveness of a vaccine against HPV in reducing the prevalence of cervical cancer. The vaccine targets the two most common types of cancer-causing HPV, HPV-16 and HPV-18, and was effective against 95% of persistent HPV infections among the study participants who received at least one dose of the vaccine.

It also demonstrated efficacy against 93% of abnormal Pap tests associated with HPV 16/18 infections and protected against cervical cancer with 100% efficacy in cases of precancerous lesions of the cervical tissue.

Focus on Viral Vectors and Vaccines

The 11th annual “Viral Vectors & Vaccines Conference,” presented by the Williamsburg BioProcessing Foundation and held recently in Williamsburg, VA, featured presentations and discussions on a range of vaccine strategies and the viral vector constructs in development for vaccine and gene therapy applications.

Vivalis’ (Nantes, France) EBx platform relies on chicken embryonic stem cells as a substrate for the production of viral vectors and recombinant proteins. Instead of infecting chicken eggs with a virus of interest, Vivalis infects chicken embryonic stem (ES) cells, which are genetically stable and immortal. A key challenge lies in culturing the ES cells without the end result being cell differentiation or cell death.

The company isolates and expands ES cells in a culture of mouse feeder cells containing a cocktail of growth factors, and adapts the cells to grow as either adherent or suspension cell cultures. Vivalis has shown that these cells express ES cell-specific markers and high levels of telomerase, are genetically stable, and are not tumorigenic. They can be grown in serum-free media at 37C.

Vivalis scaled up its process to 3-L and 7-L volumes in bioreactors with cell densities >3 million cells/mL. The company demonstrated the ability of avian and human flu viruses to infect the ES cells and produce vaccine isolates in suspension cells. Vivalis plans to file a Biologics Master File (BMF) in mid-2005 and currently has its EBx technology under evaluation for more than 15 different vaccines with vaccine producers worldwide.

In the fall, JRH Biosciences (Lenexa, KS) and Vivalis announced an R&D collaboration aimed at developing serum-free media designed for growing EBx avian cell lines.

Progress in HIV Vaccines

With 2.53.5 million estimated deaths worldwide from HIV/AIDS, the vast majority of those occurring in sub-Saharan Africa, the AIDS epidemic and HIV infection represent an obvious and urgent target for vaccine development.

John Henessey, Jr., Ph.D., senior director of bioprocess and bioanalytical research at Merck Research Laboratories (Whitehouse Station, NJ) identified the three major challenges for HIV vaccine R&D: the diversity of the target population; the diversity in amino acid sequence between different clades of the HIV virus; and issues related to vaccine supply, demand, and delivery.

The Merck HIV vaccine is a replication-incompetent adenovirus type 5 construct that is E1 deficient and propagated in an E1-complemented PER.C6 cell line. It incorporates transgenes for gag, pol, and nef, which produce the corresponding proteins when taken up by host cells, inducing an immune response to the virus.

Merck employs a variety of assays to measure the in vitro biological activity of a series of genetic constructs, including end-point dilution assays and standard curve assays to assess in vitro viral replication and infectivity, in vitro antigen expression assays intended to mimic in vivo activity, and an adenovirus genome quantitation assay based on quantitative PCR to detect full versus empty virus particles. Dr. Hennessey emphasized the need to define standardized assays for adenovirus-based vaccines

Bryan Butman, Ph.D., vp of quality at GenVec (Gaithersburg, MD) described his company’s work to develop a multiclade adenoviral vector-based vaccine against HIV. GenVec is working with the Vaccine Research Center of the NIH to create novel adenoviral vectors that encode for multiclade and multigene HIV antigens.

The company combined four different adenoviral vectors to make one tetravalent vaccine; it includes the gag and pol genes representing clade B and pg140 genes from clades A, B, and C. The expression cassette lacks E1 and E2, and E3 has been partially deleted. The complementing 293-ORF6 cell line leads to the production of replication-deficient vectors that encode for a fusion protein representing the multigene products.

GenVec is developing a scalable bioreactor system for vector production in serum-free suspension cultures with column chromatographic purification. Dr. Butman described a series of potency and identity assays used to characterize the vectors and the viral particle concentration. These include immunofluorescent assays, a transgene expression Western blot, PCR identity studies, and full-length sequencing.

Quality studies included reverse-phase HPLC of the blended product and are supported by safety and stability cell testing, aging studies, and tumorigenicity and oncogenicity studies. The HIV adenoviral vector induces a cellular immune response in cynomolgus macaques.

The Vaccine Research Center submitted an IND for clinical testing to assess the efficacy of a single administration of the vaccine at three dosing levels. GenVec has produced four bulk vaccine lots, which were quality-tested and released, and a Phase I dose-escalation clinical trial in humans began in July 2004.

The long-term goal is to assess the immunogenicity of the blended adenoviral HIV vector alone and in combination with a DNA prime-boost strategy. Challenges include the need for an efficacy trial, a more readily scalable manufacturing process, correlates of protection, quantitative potency assays for the transgene, and formulations that would allow storage of the vaccine at temperatures above -20C.

Boro Dropulic, Ph.D., founder and CSO of VIRxSYS (Gaithersburg, MD), presented the company’s work using lentiviral-based vectors for HIV gene therapy. VRX496 is the firm’s lentiviral vector expressing a 937-base antisense insert that targets the HIV envelope protein.

The goal of this approach is to transduce T lymphocytes in HIV-infected patients with the antisense payload, which then binds and destroys HIV RNA in T cells, thereby minimizing HIV replication to levels not associated with AIDS.

The company reported greater than 94% transduction of T lymphocytes in preclinical studies and greater than 100-fold inhibition of HIV replication and is currently completing a Phase I safety trial in HIV-infected patients who have failed at least two regimens of highly active anti-retroviral therapy (HAART).

Preliminary findings indicate no serious adverse events, no evidence of replication competent lentivirus, and steady CD4 counts in all patients. Two of the five patients had viral loads below baseline levels at six months post dosing, two maintained baseline viral loads, and the fifth patient had not yet undergone monitoring.

Flu: What Went Wrong?

In 2002, Lester M. Crawford, DVM, Ph.D., acting commissioner of Food and Drugs at the FDA, told a Congressional committee that the number of influenza vaccine manufacturers in the U.S. was shrinking to precarious numbersfrom 20 fifteen years previously to 3 in 2002, and to a predicted 1 supplier by 2004.

Dr. Crawford recounted that testimony at the conference and concluded that there has been little innovation and few incentives for the vaccine industry over the years, and that there is, in fact, only one U.S. supplier of flu vaccine. There are only six major manufacturers of influenza vaccine worldwide at present.

To prevent this year’s “tragedy” of too little flu vaccine, the FDA asked in 2002 for a $100 million package to support both intramural and extramural research to be carried out through the NIH. Congress answered with $50 million aimed at the development of cell culture and tissue culture-based flu vaccine research. The other $50 million was to have supported novel approaches to vaccine development.

In response to this year’s dilemma, the FDA plans to pursue dialogues with potential international partners, including those in Russia and China, and is reviewing the problems that led to the temporary closing of Chiron’s U.K. vaccine manufacturing facility and the destruction of all of the flu vaccine the company was to supply to the U.S.

In the meantime, the FDA has been able to obtain five million more vaccine doses from suppliers in Germany and Canada, and MedImmune (Gaithersburg, MD) tripled production of FluMist, its nasal spray vaccine formulation, to three million doses.

FluMist is only approved for use in healthy people aged 549 years. The FDA also performed an accelerated review of Becton-Dickinson’s (Franklin Lakes, NJ) syringe product SoloShot and approved the syringe for use. It extracts 10% more vaccine from each dose dispensed.

If a U.S. company were to enter the flu vaccine market today, it would take at least three years to bring a product to market, according to Dr. Crawford. “If the U.S. tries to do this alone, we will fail repeatedly,” he said. The bottom line: flu vaccine production needs to move away from a reliance on chicken eggs and the long timeline and costly manufacturing processes of current production methods.

Influenza Hemagglutinin

Vaxin’s (Birmingham, AL) goal is to develop a flu vaccine amenable to rapid production and administration by nonmedical personnel via intranasal or topical delivery of replication-deficient adenovirus vectors encoding influenza hemagglutinin (HA).

Construction of a recombinant adenoviral vector carrying the influenza HA gene takes about a month and eliminates the need to grow virus in chicken eggs. The design of Vaxin’s epicutaneous vaccine involves applying a skin patch containing the vaccine.

A clinical trial with an adenoviral vaccine containing the PR8 HA gene showed serum HA-inhibition antibody titers elicited with both delivery routes, but greater effectiveness with an intranasal vaccine. De-chu Tang, Ph.D., CTO at Vaxin presented studies demonstrating the ability of an adenoviral vector-based nasal influenza vaccine to protect mice against influenza.

He explained that repeat immunization with an adenoviral vaccine is not affected by pre-existing neutralizing antibodies to adenovirus, as has been problematic with adenovirus-based gene therapy constructs, because unlike in gene therapy where anti-adenoviral immunity reduces gene expression and therefore therapeutic protein production, blocking a biological effect with an adenoviral vaccine can be effective as long as enough antigen is expressed to induce an immune response.

Dr. Tang is confident that the vaccine could be given for multiple cycles without any loss in potency.

Targeting Cancer

Therion Biologics (Cambridge, MA) is developing poxvirus vector technology for use in immunotherapeutic cancer vaccines. More than 700 patients have been treated in about 30 clinical trials resulting from the company’s two lead clinical programs: PanVac-VF for pancreatic cancer (in Phase III testing); and ProstVac-VF for prostate cancer (in Phase II studies).

PanVac-VF consists of two vectors, a prime and a booster, representing two tumor antigens, CEA and MUC-1, and three costimulatory molecules. It induces a cytotoxic T lymphocyte-directed response.

David Lau, Ph.D., director of process development at Therion, described the company’s poxvirus technology platform, which begins with engineering tumor antigens into a plasmid, then integrating the antigens into the poxvirus genome, creating seed stock.

In parallel, the company then produces product lots in roller bottles and performs process development studies to optimize process parameters. The process had evolved to incorporate serum-free media, a depth filtration step, benzonase digestion, and tangential flow filtration.

In response to the threat of bioterrorism, Incell (San Antonio, TX) expanded its infectious disease program to include vaccines such as a smallpox vaccine, with an emphasis on oral and other noninjection delivery methods.

With worker safety during the manufacturing process a top priority, Incell infects cells with vaccinia virus (IMVA) and then cultures and processes the infected cells in closed systems, formulating the vaccine product for oral delivery.

The company has shown that its oral smallpox vaccine, containing live attenuated vaccinia virus, protects mice against lethal vaccinia challenge. Animals mount both humoral and cell-mediated responses to the vaccine. Incell plans to begin clinical trials in early 2005.

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