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Researchers Publish Results Using Protein Arrays to Investigate HIV-Antibody Interactions


NEW YORK (GenomeWeb) – A research team from the University of California, Irvine has published a novel technique for investigating the interactions of HIV envelope antibodies using protein microarrays.

According to the authors, the array approach could allow the same sensitivity as previously used techniques, such as ELISA, immunoblot assays, and flow cytometry, but with higher throughput and efficiency.

In the proof-of-principle study, published last week in PLoS One, the group described its approach — a prototype microarray chip comprising the HIV-1 recombinant proteins gp140, gp120, gp41, as well as several membrane proximal external region peptides — and compared their initial results using the chip to ELISA.

According to the study authors, identifying and isolating broadly neutralizing antibodies has been the major focus in HIV-1 research over the last few decades, and the follow-up analysis of how these antibodies interact with HIV has been somewhat stymied by a need for higher throughput methods.

Johannes Gach, the study's senior author, told GenomeWeb in an email this week that the bottleneck of many previous approaches for studying HIV envelope protein-antibody interactions has been the limited number of samples that can be screened at a time. "That's why we decided to use the microarray chip technology," he wrote.

The envelope glycoproteins gp41 and gp120, located on HIV's surface, represent major targets for antibody recognition, and evaluating the antibody-binding characteristics for both neutralizing and also non-neutralizing antibodies that target these proteins is an important ongoing goal in studying the disease.

In their study, Gach and his colleagues set out to create a strategy to evaluate HIV-1 specific monoclonal antibodies against 15 HIV-1 multi-clade envelope proteins and gp41 MPER analogs, spotted at three different concentrations onto a microarray chip.

Applying their array method to a set of 15 HIV-1 antibodies, the investigators saw substantial differences in the binding patters of the tested molecules. Importantly, they also found that the microarray method correlated very significantly with ELISA results — with a P value of less than 0.0001.

While there was sometimes lower correlation between the ELISA and array results — most likely due to a combination of antigen accessibility and protein stability over time — the authors concluded that with further optimization of the technology, the approach could be "an excellent tool for comprehensive antibody profiling and characterization in a high-throughput manner."

In their proof of concept, the team also showed that the approach could successfully screen the humoral immune response of human serum samples from vaccinated or HIV infected individuals, and also characterize newly identified antibodies against HIV, Gach said in his email.

Analyzing the results, the researchers were also able to identify a pattern in which individuals with higher antibody responses against HIV showed a better capacity to inactivate the virus, Gach explained.

The investigators used proteins in their initial array design because they found that correct folding and glycosylation of the envelope proteins is crucial for antibody recognition. However, Gach wrote that arraying overlapping peptides may also be an effective method for investigating linear epitopes, such as the membrane proximal external region within the gp41 glycoprotein, which is a target for several potent and broadly neutralizing antibodies and is of high interest for vaccine design against HIV.

Moving forward, Gach said that the team is working on its next prototype chip, using "state-of-the-art" HIV envelope proteins trimers generated and provided by Cornell University's John Moore. Using these trimers should allow the team to discriminate between neutralizing and non neutralizing antibody responses, he wrote.

Combining this with the group's current chip would allow a strategy where researchers could couple evaluation of HIV-1-specific antibody profiles among vaccinated individuals from clinical trials with the differentiation of neutralizing versus non-neutralizing immune responses.

Finally, Gach said, he and his team are also interested in developing a method to introduce point mutations into the SOSIP trimers in order to also map antibodies with unknown binding epitopes.

Besides HIV, he said the group is also working on developing an influenza chip using similar approaches in collaboration with Florian Krammer at the Mount Sinai Medical School.