New neutralising antibodies discovered
Richard Jefferys, TAG
A paper just published online by Science Express reports the discovery of two new antibodies capable of neutralising a broad array of diverse HIV strains. The antibodies interact with a novel conserved region of the virus envelope that is different from the sites targeted by previously described neutralising antibodies.
The research represents the first fruits of a major undertaking initiated by the International AIDS Vaccine Initiative (IAVI) in collaboration with the Scripps Institute, the Bill & Melinda Gates Foundation, Monogram Biosciences, Theraclone Sciences, a slew of scientists and over 1,800 HIV-positive volunteers who donated blood. Perhaps in keeping with the view of some sceptics that the design of an antibody-based HIV vaccine may be a mission impossible, the project goes by the espionage-invoking name of Protocol G.
The first inkling of progress came in a paper published a couple of months ago in the Journal of Virology, which appeared with little fanfare (abstract link below). A group of scientists led by Melissa Simek at IAVI described the identification of several plasma samples with broad neutralising activity using a new high throughput neutralisation assay developed by Monogram Biosciences. The assay measures the ability of antibodies to neutralise a panel of pseudoviruses that are capable of just a single round of infection. The pseudoviruses consist of a clone of the HIV genome containing a firefly luciferase gene that emits light, into which different envelope genes from primary HIV isolates are inserted. The extent to which antibodies (or plasma samples containing antibodies) prevent the various pseudoviruses from infecting susceptible target cells is measured by quantifying the amount of light emitted by the cells.
A total of 1,798 samples from HIV-positive individuals in Australia, UK, Rwanda, Kenya, Uganda, Zambia, Ivory Coast, Thailand, South Africa and the US were evaluated in the initial study. Around 1% of the samples were found to have broad neutralising activity against a panel of pseudoviruses containing envelopes from multiple different HIV isolates from clades A, B, C, D and several circulating recombinant forms including CRF01-AE.
The new Science paper focuses on just one African individual whose plasma sample was among those capable of broad neutralisation. In order to find the antibodies that were responsible for the activity, the researchers had to go fishing for the B cells that were producing them. This daunting task involved the careful characterisation of 30,300 B cells, which were spread across 23,328 tiny wells in lab dishes such that each well had just 1-2 (average 1.3) B cells in it. The B cells were given eight days to pump their antibodies into the wells, then the antibodies were taken from each and tested to see whether they bound to immobilised HIV envelope proteins (gp120 or gp41) or were able to neutralise pseudoviruses in the Monogram Biosciences assay described previously.
When the wells containing antibodies capable of the broadest and most potent neutralisation were identified, the researchers extracted the antibody-encoding sections of DNA from the B cells. The process requires extraction of two sections of B cell DNA, one responsible for producing a part of the antibody called the light chain and the other for the part of the antibody called the heavy chain. The isolated DNA sections were inserted into a laboratory cell line (293 cells) which then started churning out the antibodies encoded by the DNA, allowing researchers to figure out which DNA code was making the antibodies they were looking for by testing the antibodies for neutralisation in the Monogram assay. For the wells that contained more than one B cell, multiple light and heavy chain DNA sections were extracted and inserted into 293 cells in all possible combinations, facilitating the identification of the light/heavy chain DNA combination responsible for making the antibody of interest
The ultimate result of this staggering amount of work was the identification of two antibodies, named PG9 and PG16, with broad and potent neutralising activity. PG9 neutralised 127 out of a panel of 162 pseudoviruses containing a diverse range of HIV envelopes and PG16 neutralised 119 pseudoviruses out of the same panel. The potency of neutralisation often exceeded that of the four known broadly neutralising antibodies that were used as controls (b12, 2G12, 2F5, and 4E10), meaning that lower concentrations of PG9 and PG16 could mediate equally strong neutralisation.
While PG9 and PG16 were very effective in the neutralisation assay, they did not efficiently bind to the immobilised HIV envelope proteins that were used as part of the screening process. The researchers conclude that this is because the individual proteins do not maintain the same shape or conformation that they have when present on an intact virus, where they combine in triplicate to form what is called an envelope trimer.
The discovery of PG9 and PG16 may be important for several reasons.
It offers compelling validation of the Protocol G approach to seeking effective new antibodies, and suggests that many more are likely to be discovered. The work has been described as a tour de force, and that almost seems like an understatement.
The results indicate that although HIVs envelope is notoriously mutable, there are conserved regions of the trimer that are susceptible to antibody attack.
The potency of neutralisation suggests that if a vaccine could induce similar antibodies, they could be protective against HIV infection at concentrations known to be achievable with vaccination.
There are potential caveats however. It is unclear whether the relatively rare detection of broadly neutralising antibodies is related to specific genetic traits of the individuals they have been isolated from. If B cells from most people are not capable of making similar antibodies, then the applicability to vaccination will be limited. Researchers have also long been attempting to build mimics of HIVs native envelope trimer, and it has proven to be a considerable challenge; results to date are reminiscent of trying to bake a soufflé only to have it collapse within moments of removing it from the oven. Nevertheless, the discovery of PG9 and PG16 is likely to send scientists working on the problem scurrying back into the kitchen.
Source: TAG Basic Science web log (04 September 09)
Ref: Walker LM et al. Broad and potent neutralising antibodies from an African donor reveal a new HIV-1 vaccine target. Science, doi: 10.1126/science.1178746. Published online 3 September 2009.