Quick method for making human monoclonal flu antibodies discovered

Seeking a way to produce vaccines that better enhance immune responses, a group of researchers backed by the NIH has found a way to rapidly produce human monoclonal flu antibodies, according to a recent report.

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ATLANTA—Seeking a way to produce vaccines that better enhance immune responses, a group of researchers backed by the NIH has found a way to rapidly produce human monoclonal flu antibodies, according to a recent report.

Using cells drawn from volunteers inoculated with a seasonal influenza vaccine, the investigators made influenza-specific mAbs—monospecific, identical, infection-fighting proteins produced in large quantities—in just a few weeks rather than the typical two to three months.

The new technique could potentially be used to rapidly create mAbs for a range of uses, according to the research team, led by Drs. Rafi Ahmed and Jens Wrammert of the Emory University School of Medicine's Vaccine Center here. Also collaborating with the Emory team were Drs. Patrick Wilson and J. Donald Capra and their colleagues from the Oklahoma Medical Research Foundation in Oklahoma City. The research was supported by the NIH's National Institute of Allergy and Infectious Diseases (NIAID) and National Center for Research Resources (NCRR).

"Conventional wisdom holds that the level of pre-formed antibody is the main correlate of protection against influenza virus. However, our results, showing the rapidity of the antibody response after vaccination and the high affinity of the antibodies produced, strongly suggest that the recall response could also play a role in protective immunity," the researchers wrote. "This antibody would not, of course, prevent initial infection but could play a crucial role in preventing the spread of virus and bringing about faster resolution of the infection."

To make the new influenza mAbs, the researchers first inoculated volunteers with seasonal influenza vaccine. The scientists wanted to know if a subset of immune system cells called antibody-secreting plasma cells (ASCs) could serve as a source of mAbs.

"To generate the antibodies we used flow cytometry to isolate single vaccine-induced ASCs from which we used a multiplex single-cell RT-PCR to isolate cDNAs for the antibody heavy and light chain genes," Wilson explains. "We then cloned these into a CMV promoter driven expression vector and transfected these vectors into 293 cells. The cells secrete the antibodies that we then test for binding, hemagglutination-inhibition, neutralization, etc."

ASC activity is swift but brief. In this study, ASC responses peaked at one week after vaccination, then dropped sharply and were barely detectable after two weeks.

"Normally, you have to wait longer for an immune response to make monocolonal antibodies, but with this technique, we were able to get a response in weeks rather than months," says Dr. Diane Post, a project officer in the NIAID's Respiratory Diseases branch.

The researchers found a way to capture the fleeting ASCs that produce the initial wave of influenza-specific antibodies. The scientists were surprised to find that as many as 80 percent of the purified ASCs produced influenza-specific antibodies.

"Because so many of the ASC-generated antibodies are specific, this is a much faster way to get fully human mAbs against anything to which we have an immune response or are vaccinated against," Wilson says. "Because we were able for the first time to generate so many antibodies so rapidly, we were able to show that there is little evidence for original-antigenic sin in healthy people after vaccination. That is, the antibodies were most specific for the current vaccine strains not recycled from immune responses to previous vaccines."

With just a few tablespoons of blood, scientists can now rapidly generate mAbs that potentially could be used for diagnosis and treatment of newly emerging strains of influenza, the researchers say.

"One way this technique can be applied is if some of the antiviral resistance mutations that are detected in current therapeutics are not effective," Post says, although she points out that there is still a lot of research to be done before the technique is actually approved for use in humans.

The technique developed by the Emory and Oklahoma scientists is not limited to the production of mAbs for influenza, and the team is currently working to make mAbs for other disease agents. While the mAbs made in this study were not tested on influenza virus strains with pandemic potential, the team believes the ability to make high-affinity influenza mAbs quickly raises the possibility of deploying them in combination with other disease control strategies in the event of a global influenza pandemic. The team plans to use their technique to generate mAbs against the H5N1 subtype that causes avian flu.

"I think the strategy for generating monoclonal [antibodies] can be used to rapidly generate human mAbs to many important infectious diseases—flu, avian flu, HIC, hepatitis, anthrax, just to name a few possibilities," Wilson says. "I hope this work leads to the discovery of many new therapeutic antibodies and that diseases are really cured. I feel so strongly that this approach will be successful that I really can't wait to see what the future holds."

The study, Rapid cloning of high-affinity human monoclonal antibodies against influenza virus, was published April 30 in Nature's online publication. 

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