A paper just published online by Science Express reports the discovery of two new antibodies capable of neutralizing 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 neutralizing 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-infected volunteers who donated blood. Perhaps in keeping with the view of some skeptics 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 appended below). A group of scientists led by Melissa Simek at IAVI described the identification of several plasma samples with broad neutralizing activity using a new “high throughput” neutralization assay developed by Monogram Biosciences. The assay measures the ability of antibodies to neutralize 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-infected individuals in Australia, UK, Rwanda, Kenya, Uganda, Zambia, Ivory Coast, Thailand, South Africa and the US were evaluated in the initial study (for more information on how the samples were obtained, see "UPDATE II" below). Around 1% of the samples were found to have broad neutralizing 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 neutralization. In order to find the antibodies that were responsible for the activity, the researchers - led by Laura Walker and Dennis Burton from the IAVI Neutralizing Antibody Center at Scripps - had to go fishing for the B cells that were producing them. This daunting task involved the careful characterization of 30,300 B cells, which were spread across 23,328 tiny “wells” in lab dishes such that each well contained around 1-2 B cells (average 1.3). 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 immobilized HIV envelope proteins (gp120 or gp41) or were able to neutralize pseudoviruses in the Monogram Biosciences assay described previously.
When the wells containing antibodies capable of the broadest and most potent neutralization 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 neutralization 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 neutralizing activity. PG9 neutralized 127 out of a panel of 162 pseudoviruses containing a diverse range of HIV envelopes and PG16 neutralized 119 pseudoviruses out of the same panel. The potency of neutralization often exceeded that of the four known broadly neutralizing antibodies that were used as controls (b12, 2G12, 2F5, and 4E10), meaning that lower concentrations of PG9 and PG16 could mediate equally strong neutralization.
While PG9 and PG16 were very effective in the neutralization assay, they did not efficiently bind to the immobilized 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 conformation that they have when present on an intact virus, where they combine in triplicate to form what’s 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 HIV’s envelope is notoriously mutable, there are conserved regions of the trimer that are susceptible to antibody attack.
- The potency of neutralization 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 neutralizing antibodies is related to specific genetic traits of the individuals they have been isolated from. If B cells from most people aren’t capable of making similar antibodies, then the applicability to vaccination will be limited. Researchers have also long been attempting to build mimics of HIV’s 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.
UPDATE: The LA Times published an erroneous article on the discovery entitled "Antibodies found that prevent HIV from causing severe AIDS;" to their credit, they have now added a correction. The news article by Jon Cohen in Science Magazine clearly states that the donor "did not benefit appreciably from the antibodies" meaning they did not impact disease progression. The paper proposes that these antibodies might be able to offer substantial protection against HIV infection if they could be induced by vaccination in uninfected individuals, but does not suggest they could be used therapeutically. There have been some studies involving the infusion of previously discovered broadly neutralizing antibodies as possible therapies for established HIV infection, but the antiretroviral effects have been very modest.
UPDATE II: There seems to be a lot of confusion regarding the clinical status of the people that contributed blood samples to this research. Although all samples were obtained from asymptomatic individuals, only a subset were long-term non-progressors (LTNP) and there was no correlation between the presence of broadly neutralizing antibodies and LTNP status; this is consistent with the literature showing that T cell responses and class I HLA genes (which are linked to CD8 T cell responses) play a more important role in non-progression than antibody responses. The confusion partly stems from the use of the term "elite neutralizer" by these researchers to describe the individuals whose plasma was capable of broad neutralization of multiple HIV variants in vitro; inevitably, this has become confused with the term "elite controller" which is used to describe individuals who control their viral loads to <50 copies in the absence of treatment.
For anyone interested in the full background on the samples used in this research, this is a direct quote from the Materials & Methods section of the J. Virology paper:
"Eligible participants were age 18 years or older, were HIV-1
infected for at least 3 years prior to the day of screening, were clinically
asymptomatic, without evidence of progression to AIDS based on WHO stage III or
IV criteria or a CD4 count of <200 cells/mm3, and were not on antiretroviral
therapy (ART) for at least the previous 1 year. In Rwanda, Zambia, and the
United States, previously collected specimens from volunteers who met the
eligibility criteria were included. In Australia, previously collected
specimens from a smaller population of long-term non-progressors with a ∆32CCR5
heterozygote mutation or who were infected with a ∆nef virus were also included. In
addition, the first 101 samples screened from Rwanda and Zambia were identified
as long-term non-progressors, defined in Zambia as individuals diagnosed with
HIV-1 for at least 8 years with no clinical symptoms of AIDS and in Rwanda as
individuals diagnosed with HIV-1 for at least 16 years who had CD4 counts of >500."
Science DOI: 10.1126/science.1178746
Published Online September 3, 2009
REPORTS
Laura M. Walker 1, Sanjay K. Phogat 2*, Po-Ying Chan-Hui 3, Denise Wagner 2, Pham Phung 4, Julie L. Goss 4, Terri Wrin 4, Melissa D. Simek 5, Steven Fling 1, Jennifer L. Mitcham 3, Jennifer K. Lehrman 5, Frances H. Priddy 5, Ole A. Olsen 3, Steven M. Frey 3, Phillip W. Hammond 3, Protocol G Principal Investigators , Stephen Kaminsky 2, Timothy Zamb 2, Matthew Moyle 3, Wayne C. Koff 5, Pascal Poignard 1, Dennis R. Burton 6
1 Department of Immunology and Microbial Science, and IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA 92037, USA.
2 Design Lab, International AIDS Vaccine Initiative, New York, NY 11226, USA.
3 Theraclone Sciences, Seattle, WA 98104, USA.
4 Monogram Biosciences, Inc., South San Francisco, CA 94080, USA.
5 International AIDS Vaccine Initiative, New York, NY 10038, USA.
6 Department of Immunology and Microbial Science, and IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA 92037, USA.; Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Techonology, and Harvard, Boston, MA 02114, USA.
Protocol G Principal Investigators are listed at the end of the paper.
Broadly neutralizing antibodies (bNAbs), which develop over time in some HIV-1–infected individuals, define critical epitopes for HIV vaccine design. Using a systematic approach, we have examined neutralization breadth in the sera of about 1800 HIV-1–infected individuals, primarily infected with non–clade B viruses, and have selected donors for monoclonal antibody (mAb) generation. We then used a high-throughput neutralization screen of antibody-containing culture supernatants from approximately 30,000 activated memory B cells from a clade A–infected African donor to isolate two potent mAbs that target a broadly neutralizing epitope. This epitope is preferentially expressed on trimeric Envelope protein and spans conserved regions of variable loops of the gp120 subunit. The results provide a framework for the design of new vaccine candidates for the elicitation of bNAb responses.
Note: access to the supplementary materials & methods, which includes a graphic representation of the target of the antibodies, is free of charge.
Science 4 September 2009: Vol. 325. no. 5945, p. 1195
DOI: 10.1126/science.325_1195
NEWS OF THE WEEK
Jon Cohen
If HIV/AIDS researchers had a wish list, at the very top would sit a vaccine that could teach the body to make potent antibodies against the many strains of the virus. Despite 25 years of effort, no such vaccine is in sight, but now they are a step closer. A large team of researchers reports online in Science this week that it has identified the most powerful, broad-acting antibodies yet against multiple strains of the virus.
Journal of Virology, July 2009, p. 7337-7348, Vol. 83, No. 14
doi:10.1128/JVI.00110-09
Melissa D. Simek,1*, Wasima Rida,10, Frances H. Priddy,1 Pham Pung,2 Emily Carrow,9 Dagna S. Laufer,1 Jennifer K. Lehrman,1 Mark Boaz,1, Tony Tarragona-Fiol,17 George Miiro,5 Josephine Birungi,6 Anton Pozniak,8 Dale A. McPhee,7 Olivier Manigart,4 Etienne Karita,4 André Inwoley,11 Walter Jaoko,12 Jack DeHovitz,13 Linda-Gail Bekker,14 Punnee Pitisuttithum,15 Robert Paris,16 Laura M. Walker,3 Pascal Poignard,3 Terri Wrin,2 Patricia E. Fast,1 Dennis R. Burton,3 and Wayne C. Koff1
International AIDS Vaccine Initiative, New York, New York,1 Monogram Biosciences, Inc., South San Francisco, California,2 Department of Immunology and Microbial Science and International AIDS Vaccine Initiative Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, California,3 Rwanda Zambia HIV Research Group, Emory University, Atlanta, Georgia,4 Uganda Virus Research Institute-Medical Research Council, Entebbe, Uganda,5 Uganda Virus Research Institute-International AIDS Vaccine Initiative, Entebbe, Uganda,6 National Serology Reference Laboratory, National Center in HIV-1 Epidemiology and Clinical Research, Fitzroy, Victoria, Australia,7 St. Stephens AIDS Trust, London, United Kingdom,8 Advanced BioAdjuvants, LLC, Omaha, Nebraska,9 Arlington, Virginia,10 Diagnostic Center and Research on AIDS and Opportunistic Diseases, CeDReS, Abidjan, Ivory Coast,11 Kenya AIDS Vaccine Initiative, University of Nairobi, Nairobi, Kenya,12 Department of Preventive Medicine, SUNY-Downstate Medical Center, Brooklyn, New York,13 Desmond Tutu HIV-1 Foundation, University of Cape Town, Cape Town, South Africa,14 Department of Clinical Tropical Medicine, Clinical Infectious Disease Research Unit, Vaccine Trial Center, Mahidol University, Bangkok, Thailand,15 Department of Retrovirology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand,16 IAVI Core Laboratory, Imperial College London, South Kensington, London, United Kingdom,17
The development of a rapid and efficient system to identify human immunodeficiency virus type 1 (HIV-1)-infected individuals with broad and potent HIV-1-specific neutralizing antibody responses is an important step toward the discovery of critical neutralization targets for rational AIDS vaccine design. In this study, samples from HIV-1-infected volunteers from diverse epidemiological regions were screened for neutralization responses using pseudovirus panels composed of clades A, B, C, and D and circulating recombinant forms (CRFs). Initially, 463 serum and plasma samples from Australia, Rwanda, Uganda, the United Kingdom, and Zambia were screened to explore neutralization patterns and selection ranking algorithms. Samples were identified that neutralized representative isolates from at least four clade/CRF groups with titers above prespecified thresholds and ranked based on a weighted average of their log-transformed neutralization titers. Linear regression methods selected a five-pseudovirus subset, representing clades A, B, and C and one CRF01_AE, that could identify top-ranking samples with 50% inhibitory concentration (IC50) neutralization titers of 100 to multiple isolates within at least four clade groups. This reduced panel was then used to screen 1,234 new samples from the Ivory Coast, Kenya, South Africa, Thailand, and the United States, and 1% were identified as elite neutralizers. Elite activity is defined as the ability to neutralize, on average, more than one pseudovirus at an IC50 titer of 300 within a clade group and across at least four clade groups. These elite neutralizers provide promising starting material for the isolation of broadly neutralizing monoclonal antibodies to assist in HIV-1 vaccine design.
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