HIV researchers of many stripes convene at the annual Keystone meeting and discuss some familiar obstacles
By Kristen Jill Kresge and Simon Noble
The Whistler-Blackcomb Mountains form one of the world's premier ski resorts, the largest in North America, and provided the backdrop for the 2007 Keystone Joint Symposia on HIV Vaccines: From Basic Research to Clinical Trials and Molecular and Cellular Determinants of HIV Pathogenesis. Whistler got its name from the whistling calls of the marmots that populate the alpine area, and this year's conference did have something of a 'Groundhog Day' feel to it—the same familiar scientific obstacles and a field waiting for that vital breakthrough.
There are now more than 30 ongoing clinical trials of AIDS vaccine candidates but one thing they all have in common is that none are expected to induce the much sought after protective antibody response capable of neutralizing a diverse array of HIV isolates. The consensus among most researchers is that, at best, the cellular-mediated immunity (CMI)-based candidates might significantly lower the viral load in vaccinated individuals who do become HIV infected, delaying disease progression and reducing the risk of transmission.
This would be no small accomplishment—such a first generation, partially-effective AIDS vaccine would help curtail the global epidemic and perhaps reduce the number of years that an HIV-infected person has to take antiretrovirals (ARVs). But just what researchers can expect from this pipeline of candidates is still unclear and may remain so until results are available from the first test-of-concept trials with CMI-based vaccines in a few years time.
Merck expects results from one of their first Phase IIb trials with an adenovirus serotype 5 (Ad5) candidate vaccine at the end of 2008 or early 2009. In 2011 the Vaccine Research Center (VRC) at the US National Institute of Allergy and Infectious Diseases (NIAID) anticipates having results from their soon to begin test-of-concept trial known as PAVE 100, which will administer DNA and Ad5 candidates as a prime-boost. Even then, as some researchers point out, there is little agreement about what the parameters are for success of these vaccines. Bruce Walker of Massachusetts General Hospital urged the field to come to a consensus regarding what level of reduction in viremia would constitute success with a CMI-based vaccine.
In the meantime, AIDS vaccine researchers are intensifying efforts into fundamental immunological questions rather than advancing too many new CMI-based candidates into clinical trials. Jonathan Yewdell of NIAID gave a highly entertaining and thought-provoking talk on the first evening of the conference, starting out by emphasizing the truism that there are still many unknowns in immunology and plenty of scope for much more basic research. His research group is initiating studies in neuroimmunology—an intriguing field he admitted "doesn't have the best reputation"—looking at the involvement of sympathetic innervation on antiviral immunity. They have found that CD8+ and CD4+ T-cell responses can be increased up to 4-fold by ablating sympathetic neurons.
Yewdell then switched to a topic that he and colleagues Jack Bennink and Luis Anton have pioneered. Class I peptide complexes can reach sufficient levels to trigger CD8+ T-cell activation within an hour of adding virus to cell culture. Since these viral proteins must compete with a pool of billions of already translated cellular proteins, this suggests that there must be an active sampling mechanism in place to direct such a rapid presentation process. He and colleagues hypothesize that the peptides presented by MHC class I molecules originate from defective ribosomal products (DRiPs) that arise from imperfections in transcription, translation, post-translational modifications, or protein folding, and are trying to define the contribution to antigen processing. Provocatively he declared, only partly tongue in cheek, "we're not here to refine our favorite model to the nth degree, we're here to overturn the applecart."
In its latest incarnation, the DRiP hypothesis further postulates that there is a subset of ribosomes that purposely generates DRiPs for antigen presentation; the 'immunoribosome.' Yewdell pointed out in his talk that protein translation is much more intricate than generally appreciated, that different mRNAs and different tissues use different tRNAs, and that 451 tRNAs have been annotated in the human genome. He also pointed out that the largest locus for tRNAs is in the HLA complex, suggesting that the immune system and viruses probably exploit tRNA heterogeneity, and presented evidence that virus infections induce misloading of tRNAs with the wrong amino acid, perhaps to better generate DRiPs. He ended by predicting to the audience that "tRNAs are in your future."
Program updates: CHAVI
A new development at this year's Keystone Symposia was a session of HIV Vaccine Program Updates that gave representatives from some of the major research programs the opportunity to describe their respective approaches and progress. Bart Haynes of Duke University kicked off this session with an update on the progress of the Center for HIV/AIDS Vaccine Immunology (CHAVI), of which he is director. The CHAVI Grant was awarded by NIAID in July 2005, and Haynes began by giving an overview of the infrastructure that has been put in place that, as of March 2007, includes 94 investigators at 51 institutions or sites in Africa, Europe, and the US.
He then gave an update on the progress of three of CHAVI's Discovery Teams, beginning with the Genetics Discovery Team that is looking genome-wide for human gene variants that affect early virus set-point. They are using a microarray chip that can check 550,000 single-nucleotide polymorphisms (SNPs) to study their EuroCHAVI Consortium cohorts in five European countries and Australia. Preliminary results from these studies have confirmed the strong link between the HLA*B5701 allele and low virus set-point, calculating that this allele accounts for 8% of the total variability in set-point. The team has found other polymorphisms associated with set-point, two of which are genome-wide statistically significant, and are now further evaluating these SNPs in additional cohorts.
Another Discovery Team is conducting studies in patients with acute HIV-1 infection in cohorts in South Africa and the US to look for signatures and functional characteristics of transmitted viruses to eventually develop new immunogens. The analysis strategy is to align over 4000 B-clade env sequences from 103 acutely-infected individuals to identify transmission signatures, and Haynes reported on two initial signatures in the env leader sequence and gp120. Functional and immunogenicity studies are underway.
The B Cell Discovery Team is looking to define viral and host mechanisms that affect antibody production during acute HIV-1 infection. Haynes presented some ideas as to what might account for the delay between the initial infectious event and the peak antibody titers against Env, and hypothesized that some species of broadly neutralizing antibody may be under the control of B-cell immunoregulatory events. Published evidence indicates that there is also massive apoptosis occurring during HIV infection, and the team has now looked at apoptosis during the time of viral load ramp-up in acute HIV infection. They do indeed see elevated levels of apoptosis markers (FasL, TNFRII, and TRAIL) in plasma that is temporally associated with the initial viral load increase, and postulate that this may mediate immune suppression of early B-cell responses.
Program updates: IAVI
Next up was Wayne Koff, Senior Vice President of R&D at IAVI, who said the theme of his presentation would be fostering innovation in AIDS vaccine discovery and development. The scientific priorities for IAVI are to design, develop and advance to efficacy trials vaccine candidates that (i) elicit broadly neutralizing antibodies against HIV or (ii) control HIV infection to a similar degree that live attenuated SIV protects against pathogenic SIV challenge. IAVI has three main scientific consortia working on these goals—Neutralizing Antibody Consortium (NAC), Vector Design Consortium (VEC), Control of HIV/SIV-Live Attenuated Consortium (LAC)—in addition to the IAVI Vaccine Development Laboratory at State University New York, Brooklyn, and the IAVI Human Immunology Laboratory at Imperial College, London, as well as a product development infrastructure and a network of partner sites in developing countries.
Koff first talked about the NAC and the neutralizing antibody problem. In designing immunogens to elicit broadly-neutralizing antibodies, one of the current major roadblocks is screening candidates for immunogenicity. IAVI has been using large-scale automated neutralization assays with pseudotyped virus for some time, and is also now conducting a study called Protocol G, a large-scale screen of sera looking for broadly-neutralizing monoclonal antibodies (MAbs). Newly-identified MAbs will then enter the new robotic platform at The Scripps Research Institute to accelerate crystallization and structural studies that will in turn inform immunogen design.
Koff briefly talked about the Control of HIV/SIV-LAC, specifically identifying antigenic targets for HIV control in human elite controllers who spontaneously control their HIV infection below undetectable levels, as well as some exciting data coming out of the LAC with SIVmac239Δnef (see below, Watkins).
The various research groups that make up the VEC are now developing novel virus vectors that seek to induce persistent immunity—cytomegalovirus (CMV; see below, Picker), reovirus, and paramyxovirus—or chimeric virus vectors that mimic the live attenuated SIV vaccines—Venezuelan equine encephalitis virus/HIV, and vesicular stomatitis virus/HIV.
With regard to clinical trials, Koff reported that in addition to 13 completed AIDS vaccine clinical trials and 4 ongoing, IAVI also has a series of clinical research studies completed, underway, or in development to test everything from HIV prevalence and incidence to acute infection and vector seroprevalence. One of these he described in some detail, Protocol D in 2400 participants in Uganda, Kenya, Zambia, and Rwanda to determine reference ranges for a number of clinical parameters relevant to the immunological and general health status of trial volunteers in developing countries. He presented data on eosinophil and neutrophil counts and showed that these values deviated extensively from the ranges found—and currently universally used in clinical trials—in 'Western' populations. These datasets will be published this year in a series of papers.
IAVI is collaborating with the HIV Vaccine Trials Network (HVTN) and US Military HIV Research Program (USMHRP) in the ongoing Phase I/II multi-site evaluation of the VRC DNA and Ad5 AIDS vaccine candidate. IAVI is conducting the trials at sites in Rwanda and Kenya, and Koff presented data showing that 72% of vaccinees given 1010 particles responded by interferon (IFN)-gELISPOT assays. When segregated by pre-existing anti-Ad5 antibody titer, 78% of vaccinees with titers <1000 were responders, compared to 57% of vaccinees with higher titers. This compares favorably with the data previously obtained in relation to the Merck Ad5 AIDS vaccine candidate currently in Phase IIb trial.
In response to the potential of pre-existing immunity to Ad5 to compromise any immunogenicity or efficacy of Ad5-based candidates, IAVI has been developing and testing AIDS vaccine candidates based on adenovirus serotypes with low seroprevalence, including human Ad35 and chimpanzee Ad7 (C7), the latter in partnership with GlaxoSmithKline. In rhesus macaque immunogenicity studies Koff showed that heterologous C7 prime/Ad35 boost elicited the highest T-cell responses.
Koff finished his presentation by talking about Phase II Screening Test of Concept (STOC) trials, a novel clinical trial paradigm that would garner preliminary efficacy data on promising vaccine candidates in a short period of time and in fewer trial participants. A series of these trials could rank the most promising candidates in a relatively rapid timeframe, rather than test them all in lengthy, expensive Phase III trials in >10,000 volunteers. Koff's suggested numbers for STOC trials were 30 incident HIV infections to detect a 1 log suppression of viral load, which would require a 4% incidence and 500 subjects with 18-month post-vaccination follow up. STOC trials might also have utility in systematically testing other parameters such as different immunogen combinations in a specific platform or delivery routes.
Program updates: VRC
Gary Nabel, Director of the VRC at NIAID, then followed with a comprehensive update on the VRC multiclade AIDS vaccine candidate that Koff mentioned that is being tested in the Partnership for AIDS Vaccine Evaluation (PAVE) collaboration. Nabel began by explaining that the DNA/Ad5 prime boost platform was the most potent currently available for inducing CD8+ T cells, and that the candidates contained—in addition to the internal Gag, Pol, and Nef antigens—Env antigens because evidence suggested they could increase the breadth of immunity and potentially diminish viral escape. He presented data that indicated that the DNA/Ad5 combination elicited more polyfunctional (as measured by the number of cytokines produced) HIV-specific CD4+ and CD8+ T cells than either candidate alone, and that DNA/Ad5 but not Ad5/Ad5 showed efficacy against SIVmac239 challenge in macaques.
Nabel then talked about the PAVE 100 Phase IIb efficacy trial that will further test the VRC candidate subsequent to the Phase I/II trials currently underway. The multi-site collaboration between HVTN, USMHRP, IAVI, and the US Centers for Disease Control and Prevention (CDC) will enroll 8500 participants in North and South America, the Caribbean, and eastern and South Africa starting later this year, with results available in 2011. Nabel then went on to discuss potential outcomes and what should be done to best respond to those outcome scenarios.
If the VRC DNA/Ad5 candidate is a complete success then it will be necessary to identify the most expedient way to obtain licensure, and Nabel expects that would be no sooner than 2013, and probably more likely around 2016. Scientifically, complete success would require that immune correlates of the protection be determined to inform further improvements in vaccine design. It would also be necessary to identify pharmaceutical collaborators or licensees for large-scale manufacture, and to develop the infrastructure required to administer the vaccine to those at high risk throughout the world.
In an outcome Nabel called "success with limitations" he said a 2016 licensure would be ambitious and it would be necessary to refine the existing candidate for a licensure trial. This would entail, among other things; engineering improved T-cell immunogens to elicit better coverage against circulating isolates; optimizing DNA immunogenicity through adjuvants, electroporation delivery, and stimulatory cytokines; and developing second generation recombinant adenovirus (rAd) vectors like rAd35 (see below), hexon chimeric rAD5, and rAd26. In preparation the VRC has engineered a rAd35 prototype candidate that contains a single clade A Env insert, and an Investigational New Drug (IND) submission has been filed with the Food and Drug Administration (FDA). Contingent on its approval, Phase I studies are scheduled for May this year that will test rAd35 alone and in combination with the Ad5 and/or DNA.
In the event that PAVE 100 shows no efficacy then Nabel said it would be necessary to develop next generation vaccines, particularly candidates that elicit broadly neutralizing antibodies, mucosal and/or innate immunity, and consider the use of persistent vectors. Nabel said it may also be time to seriously consider radical new concepts like inducing responses to self molecules like CD4 or CCR5 to try to interfere with HIV infection.
Nabel finished up by describing research that the VRC is undertaking to look at the neutralizing antibody problem, such as the mutagenic stabilization of the CD4-bound conformation of gp120 to produce immunogens (see below, Wyatt).
Louis Picker of Oregon Health and Science University presented data in the rhesus macaque model on T-cell populations, particularly the elicitation of effector memory T (TEM) cells that he suspects may be in the best position, both spatially and temporally, to exploit the window of opportunity that precedes the massive replication and mutation in SIV infection. He is hoping that SIV-specific cells with TEM differentiation can be elicited with non-lentiviral vectors, specifically the novel rhesus cytomegalovirus (RhCMV) vectors that he and colleagues are developing. Picker called CMV the "quintessential" inducer of TEM responses, and other potential advantages of CMV as a vector include: efficient re-infection; low pathogenicity; lifetime persistence of both vector and response; and a large genome with potential for manipulation.
After showing that the RhCMV vectors express the SIV gene inserts at high levels, the team assessed the immunogenicity of vaccine candidates with different inserts. Picker showed data that repeated re-immunization with RhCMV containing different inserts elicited robust step-wise increases of SIV-specific CD4+ and CD8+ T-cell responses both in peripheral blood and lung lavage, a representative mucosal site, even at very low doses. He described CMV as the "Energizer Bunny" of vaccine vectors that keep going and going, resulting in responses that "rival or exceed" those elicited by any other viral vector to date. All vectors persisted at secretory sites (mouth and bladder).
Picker concluded by saying that definitive challenge studies in immunized macaques will happen later this year, and thinks that RhCMV vectors will provide the opportunity to see if "TEM flavored" T-cell responses offer enhanced efficacy against pathogenic SIV challenge. He and colleagues also plan to investigate how genomic modification might increase immunogenicity and decrease pathogenicity through deletion of non-essential genes involved in immune evasion or modulation, and the application to human CMV.
One of the most exciting new findings presented at Keystone came from David Watkins of the University of Wisconsin-Madison. As part of IAVI's LAC his research group has been looking at live attenuated SIVmac239Δnefprotection of rhesus macaques. There are several published studies where macaques have been homologously protected from pathogenic SIVmac239 challenge, reducing set-point viral load by about 1.5 logs at most.
However, rather surprisingly, very recently his group have found that animals vaccinated with SIVmac239Δnef and challenged six months later with pathogenic SIVsmE660 are robustly protected from this heterologous challenge—the geometric mean viral load four weeks after challenge was lowered by an impressive 3 logs. Macaques with Mamu-B*08 and -B*17 MHC alleles showed the most impressive control, and Watkins alluded to data that show these alleles bind similar peptides to the HIV-ameliorating human HLA-B*57 and -B*27 alleles
Watkins cautioned that it was still early days but such a reduction in viral load is the most impressive described to date with a heterologous challenge, and he is hopeful that the system will provide immunogenetic clues to further understanding heterologous control.
Lessons on cellular immunity
While cellular immune responses are unlikely to be sufficient to protect people from HIV infection, they do clearly play a role in the control of HIV in infected individuals. Walker has been studying different categories of people who spontaneously control HIV infection without ARV therapy. In his cohort of elite controllers—HIV-infected individuals who maintain a viral load below 50 HIV RNA copies/ml of blood—more than 25% of the individuals do not have any genetic characteristic, such as an HLA allele like B*57 or a particular chemokine receptor, that has been previously shown to correlate with protection against HIV. The level of neutralizing antibody responses in Walker's elite controllers are also very low and the CD8+ T-cell responses are both of lower breadth and magnitude than those seen in progressive infection, targeting primarily Gag. John Mascola of NIAID reported also seeing a greater breadth of T-cell responses in progressors than in long-term nonprogressors, in his cohort of 32 HIV-infected individuals.
Yet something permits these individuals to still keep HIV in check. "We don't really have a clue as to why this is, but we're trying to recreate this phenotype with a vaccine," said Walker. "We sure hope it's the immune system that's doing something because that's good news for vaccine development." His group is now studying the antiviral effects of cytotoxic T-lymphocyte (CTL) responses in elite controllers.
It's not magic
To further expand the body of knowledge on how cellular immunity functions in HIV, researchers are also studying the role of CD4+ and CD8+ T-cell responses in controlling or clearing other viral infections. "The way T cells behave isn't magic," said Picker, "there are rules and we're beginning to understand those rules."
Rick Koup and colleagues at the VRC are investigating how CD8+ T-cell responses are involved in vaccine-induced protection where cellular immunity is known to play an important role and also how these cells function in response to other pathogens, including vaccinia, CMV, and HIV-2 infections. His group has observed an association between polyfunctional CD8+ T cells that secrete multiple cytokines/chemokines—including CD107a, IFN-g, TNF-a, MIP-1b, and interleukin (IL)-2—and control of HIV replication. Polyfunctional CD8+ T cells secrete even more IFN-g, on a per cell basis, than a monofunctional cell that is only secreting this single cytokine, said Koup. But he also points out that while such polyfunctional CD8+ T-cell responses are capable of providing life-long protection against vaccinia virus, they aren't even able to protect against superinfection with a different strain of HIV.
In CMV infection, CD4+ T cells produce higher levels of MIP-1b than in HIV infection. Koup said studies have shown that cells that secrete MIP-1b and are CD57+ contain 20-fold lower levels of HIV, indicating this cytokine may actually play a protective role against HIV infection.
The adaptive immune system, especially the T-cell response, controls CMV for the lifetime of the host. There is evidence that this immune control is mediated, at least in part by CD4+ T cells, which can perform both their traditional helper function and antiviral effector functions, according to a presentation by David Price of Oxford University in the UK.
He is studying, in collaboration with colleagues at the VRC and Oregon Health Sciences Center, the role of CMV-specific CD4+ T cells in persistent infection of rhesus macaques. After sorting CMV-specific CD4+ T cells expressing both CD25 and CD69—markers of T-cell activation—in both acute and chronic CMV infection in macaques, Price observed that the CD4+ T-cell repertoire is polyclonal during acute infection and evolves substantially over time, becoming much less polyclonal during the period of chronic infection.
When macaques that were already infected with RhCMV were re-challenged 224 days later, there were transient breakthroughs of virus, but overall the animals controlled the virus more effectively than in primary infection. Their CD4+ T-cell responses were also more diverse and polyclonal during secondary infection. Furthermore, the dominant CD4+ T-cell clonotypes in these repertoires were a repeat of those observed in primary infection. These data indicate that the CMV-specific CD4+ T-cell clonotypes induced after primary infection persisted in the memory pool and contributed to the immune response after reinfection.
Bigger isn't always better
Charles Bangham of Imperial College London is studying the antiviral CTL response in human T-lymphotropic virus type 1 (HTLV-1), which preferentially infects both CD4+ and CD8+ T cells. This virus infects from 10 to 20 million people worldwide and can cause chronic inflammatory diseases and a rapidly progressing and fatal leukemia, but 95% of those infected are able to effectively control the virus and remain healthy, leading Bangham to ask why most people infected with HTLV-1 don't develop disease.
Individuals infected with HTLV-1 have strong virus-specific immune responses, involving both antibodies and T-cell responses. The HTLV-1-specific CTLs are extremely abundant, chronically activated, and directed primarily against the Tax protein of the virus (Curr. Opin. Immunol. 12, 397, 2000), and according to Bangham these cellular immune responses play a dominant role in controlling HTLV-1 infection.
The HTLV-1 viral load varies greatly between individuals and the main determinant of this variation in vivo is the lytic efficiency of virus-specific CD8+ T cells (CTLs). Bangham said that in a typical HTLV-1-infected individual, each CD8+ T cell kills approximately five virus-infected cells per day, leading to the destruction of almost two billion HTLV-1-infected CD4+ T cells each day.
The efficiency of these CTL responses is determined partly by HLA class 1 genotype. However Bangham said there is not an observed correlation between the frequency of virus-specific CTLs and their efficacy—many infected individuals who mount the most effective cellular immune responses also have the lowest quantity of virus-specific T cells. Rather, their lytic capacity is associated with high granzyme expression and, according to yet unpublished data from Bangham's group, Tax-negative CD4+ FoxP3+ T regulatory cells seem to control the rate of CTL killing capacity in HTLV-1-infected individuals.
This analysis predicted that CD4+ T memory cells—the main cell type infected by HTLV-1—turn over abnormally fast in HTLV-1 infection. Recently, Bangham's group verified this by tagging lymphocytes in vivo with deuterium-labeled glucose.
Bangham contends that studying the cell-mediated immune response to other viral infections, especially persistent viral infections such as HTLV-1 or CMV, will help to improve the understanding of HIV infection and can be useful to AIDS vaccine development. "It will enable us to identify and quantify the factors that determine the efficacy of the antiviral cellular immune response and to understand the dynamics of persistent viral infections," he says. The finding that the frequency of virus-specific cellular immune responses is not necessarily a reliable guide to their efficacy in HTLV-1 infection also seems to hold true for HIV infection, based on some of Walker's data from HIV controllers. In these individuals immune responses directed towards less variable regions of the virus, like Gag, are correlated with immune control even if they exist at much lower levels than CD8+ T cells that target the highly variable Env. Walker said it's not yet time to start tossing out vaccine candidates that include env, but he does suggest caution. "We need to look at this data closely and see if there is competition between Gag- and Env-focused responses," added Walker.
Picturing the Holy Grail
While researchers sort out the functions and meaning of cellular immune responses, structural biologists are painstakingly trying to figure out how and where the already-identified broadly-neutralizing antibodies bind to HIV. Several presentations focused on how structural biology can be used to identify sites of vulnerability on the virus, as was done recently by the work of Peter Kwong and colleagues at the VRC (see An Interview with Dennis Burton: Structure-function in HIV research, IAVI Report 11, 1, 2007). Information like this should eventually lead to the structural-based design of immunogens that can be used in AIDS vaccine candidates. But as Rich Wyatt of the VRC pointed out, this has been a remarkably difficult task and in the 9 years following the elucidation of the structure of HIV gp120 bound to CD4 and the CD4-induced antibody 17b, there has been only incremental progress.
Part of the reason for this difficulty is the extreme flexibility of the gp120 molecule, which hides the functionally-conserved regions from neutralizing antibodies and diverts the immune response to irrelevant conformations; it also employs other diversionary tactics, like immunodominant variable loops and a mask of glycan residues. Ideally, researchers would like to lock the gp120 protein into a particular conformation related to that of the functional spike to see if they can induce different antibodies or increase the neutralization potency of antibodies against the virus. Wyatt and his colleagues have been trying to do just this by stabilizing the CD4-bound conformation of gp120 with various space-filling mutations and internal added cysteine pairs within gp120, with the aim of eliciting antibodies to the CD4 binding site.
To try to lock the gp120 trimer into place, Wyatt's group started out by making YU2 gp120 trimers that incorporated space-filling mutations. They then measured the entropy readout of the molecule to determine the effect of these mutations on its stability. The space-filling mutations resulted in a trimer that was 30-40% stabilized, but it only minimally increased the antibody neutralization potency as compared with other pseudotypes and offered no increase in breadth of antibodies directed towards the CD4 binding site. Next they tried also adding cysteine pairs into the core of gp120 to further stabilize the trimer relative to CD4 interaction; as a by-product, 17b affinity also increases with these changes. With these additional changes, core gp120 interaction with CD4 was stabilized by 55% as measured by calorimetry, but the recombinant Env molecules would no longer fold into a trimer. Testing immunogenicity of the stabilized core proteins in rabbits revealed that 17b-like antibodies were elicited at high titer but neutralization capacity was not enhanced.
Wyatt's group will now assess the percentage of antibodies directed towards the stabilized gp120 core that are CD4-like and will also attempt to stabilize the CD4-binding region by alternative strategies. They see the large increase in 17b-affinity as establishing proof of principle and now hope to be able to do the same with the CD4 binding site.