TAG HIV Basic Science, Vaccines, and Cure Project Blog

Novel, Broadly Active HIV Inhibitor Shows Potential

A paper published yesterday in Nature has stirred considerable excitement and widespread media coverage. Led by the laboratory of Michael Farzan at The Scripps Research Institute, the research involves a newly designed inhibitor of HIV named eCD4-Ig. The inhibitor is designed to bind the HIV envelope at sites that attach to CD4 and CCR5 molecules on CD4 T cells; blocking this interaction prevents HIV from gaining entry and infecting the cell. In laboratory experiments, eCD4-Ig showed activity against an unprecedentedly broad array of HIV and SIV variants, including viruses known to be resistant to even potent neutralizing antibodies. In addition to the greater breadth, activity was generally achieved at lower concentrations than has been documented with the broadly neutralizing antibodies (bNAbs) described to date. Due to similarities between HIV envelope binding to CCR5 and CXCR4 co-receptors, eCD4-Ig also proved capable of neutralizing CXCR4-dependent virus isolates. These results prompted the researchers to explore the preventive potential of eCD4-Ig in the rhesus macaque model, and it is the outcome of this part of the study that has particularly spurred the publicity.

To deliver the eCD4-Ig (which was modified to match the rhesus macaque form of the protein), the researchers chose to incorporate the gene encoding eCD4-Ig into an AAV vector. AAV vectors are taken up by muscle tissue and act as a factory for producing the encoded protein; for this reason they have been widely used in gene therapy studies (to deliver factor IX to hemophiliacs, for example) and are also under evaluation as a method to deliver neutralizing antibodies against HIV. One small twist with the eCD4-Ig macaque study is that the researchers delivered an 80/20 mix of two AAV vectors: one encoding eCD4-Ig, the other encoding the gene for tyrosylprotein sulfotransferase 2 (TPST2). The role of the TPST2 is to alter the eCD4-Ig protein via a process called sulfation, and this is necessary to make the eCD4-Ig active. It’s not evident from the paper whether preliminary experiments indicated the addition of TPST2 was needed, or if the decision was based solely on biological considerations.

Four macaques were administered the AAV vector mix and four served as controls. All animals were subsequently challenged intravenously with escalating doses of a hybrid SIV/HIV virus (SHIV-AD8). The four controls all eventually became infected, but the AAV recipients resisted infection despite multiple challenges over a 34-week period. Serum levels of eCD4-Ig were stable at the end of the 40-week study, and sera from the macaques neutralized HIV in vitro just as effectively as eCD4-Ig produced in the laboratory. Although antibody responses against the eCD4-Ig could be detected in the macaques, the levels were very low (much lower than were seen against the broadly neutralizing monoclonal antibodies 3BNC117, NIH45-45, 10-1074 and PGT121).

The researchers cite several features of the approach that may make it suitable for advancing into human trials.

• Protection was achieved at eCD4-Ig concentrations that are likely sustainable in humans, and the challenge dose of virus was higher than is the case in most human transmission events.
• Previous macaque studies using AAV vectors to encode bNAbs have shown that protective titers can be sustained for at least several years.
• A related protein that only targeted the CD4 binding site of HIV, CD4-Ig (also known as PRO 542), has been tested in human trials and found to be safe.
• eCD4-Ig does not appear likely to provoke significant immune responses against itself in humans.

 But there are also some potential hurdles and caveats: 

• The macaque experiment involved intravenous challenges and efficacy against a mucosal challenge has yet to be assessed.
• Whether eCD4-Ig can safely be administered to humans remains to be established.
• Advancing what is essentially a gene therapy approach into healthy individuals raises safety concerns that regulatory agencies will want to scrutinize very carefully (notably, the first ever phase I human clinical trial of an AAV vector encoding a bNAb against HIV began in the UK last year).
• Further research is needed to establish if HIV can become resistant to eCD4-Ig, although the researchers believe that if the virus did develop the ability to bind CCR5 and CD4 in the presence of eCD4-Ig, the binding efficiency would be extremely impaired.
• The safety of delivering the adjunctive TPST2 protein will need to be established. Rather than use a separate AAV vector to deliver TPST2, as was the case in the macaque study, the aim is to develop a single AAV vector that encodes both eCD4-Ig and TPST2. Human TPST2 is naturally expressed in multiple tissues, including muscle, giving some reason to hope that delivery via AAV vector would not be problematic.
• There is evidence that the presence of AAV-specific memory CD8 T cell responses in humans can limit the ability of AAV vectors to deliver their protein cargo (a problem seen in hemophilia gene therapy trials). Whether the problem might affect AAV-based approaches for HIV is not yet known. Strategies to limit CD8 T cell recognition of AAV vectors are actively being pursued.  

The paper does not explicitly discuss whether eCD4-Ig might have therapeutic potential (focusing instead on prevention) but notes that it is a potent inducer of antibody-dependent cell-mediated cytotoxicity (ADCC); it has been postulated that induction of ADCC against HIV-infected cells could be a component of a combination strategy to eliminate HIV reservoirs. A press release from the National Institute of Allergy and Infectious Diseases (NIAID) addresses the issue more directly and refers to eCD4-Ig as a “potential long-acting HIV therapeutic,” with a quote from Michael Farzan stating: “if one could inject either eCD4-Ig or our gene therapy tool into people with HIV infection, it might control HIV for extended periods in the absence of antiretroviral drugs.” The release also notes that therapeutic studies in macaques are getting underway, funded under NIAID’s recent Beyond HAART: Innovative Therapies to Control HIV-1 RFA.

According to an article in the New York Times, the researchers are considering a stepwise development plan that would first test infusions of the eCD4-Ig protein in healthy volunteers, then evaluate AAV-delivered eCD4-Ig (and, presumably, TPST2) in HIV-positive individuals, then finally move the AAV-delivered version into trials in HIV-negative populations at high risk of HIV infection. In the Wall Street Journal, Farzan articulates the hope that “human trials could begin within a year” but this may be overly optimistic; given the additional work that needs to be done, two or three years might be more realistic.

Links to several news articles about the research are below.

Stopping HIV with an artificial protein - Jon Cohen, Science Magazine, February 18, 2015

New Approach to Blocking H.I.V. Raises Hopes for an AIDS Vaccine - Donald G. McNeil Jr., New York Times, February 18, 2015

Molecule Shows Ability to Block AIDS Virus - Betsy McKay, Wall Street Journal, Feb. 18, 2015

Update 3/6/15: A webcast of Michael Farzan's presentation at CROI 2015 is now available. In the talk Farzan reveals that, since the publication of the Nature paper, the eCD4-Ig recipients have been challenged with two higher doses of SHIV-AD8 and remain uninfected.

Nature (2015) doi:10.1038/nature14264

Received 29 June 2013 Accepted 27 January 2015 Published online 18 February 2015

AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges 

Matthew R. Gardner,  Lisa M. Kattenhorn,  Hema R. Kondur,  Markus von Schaewen,  Tatyana Dorfman,  Jessica J. Chiang,  Kevin G. Haworth,  Julie M. Decker,  Michael D. Alpert,  Charles C. Bailey,  Ernest S. Neale,  Christoph H. Fellinger,  Vinita R. Joshi,  Sebastian P. Fuchs,  Jose M. Martinez-Navio,  Brian D. Quinlan,  Annie Y. Yao,  Hugo Mouquet,  Jason Gorman,  Baoshan Zhang,  Pascal Poignard,  Michel C. Nussenzweig,  Dennis R. Burton,  Peter D. Kwong,  Michael Piatak    Jeffrey D. Lifson,  Guangping Gao,  Ronald C. Desrosiers,  David T. Evans,  Beatrice H. Hahn,  Alexander Ploss,  Paula M. Cannon,  Michael S. Seaman  & Michael Farzan

Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs)1, 2. However, even the best bNAbs neutralize 10–50% of HIV-1 isolates inefficiently (80% inhibitory concentration (IC80) > 5 μg ml−1), suggesting that high concentrations of these antibodies would be necessary to achieve general protection3, 4, 5, 6. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean half-maximum inhibitory concentration (IC50) < 0.05 μg ml−1). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2 and simian immunodeficiency virus isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46 and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17–77 μg ml−1 of fully functional rhesus eCD4-Ig for more than 40 weeks, and these macaques were protected from several infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well-characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.

Nature (2015) doi:10.1038/nature14205

Published online 18 February 2015

HIV: Tied down by its own receptor

Nancy L. Haigwood

Report of “Aggressive” HIV Variant in Cuba

On January 28th, a paper reporting evidence of rapid progression associated with a recently identified HIV-1 recombinant circulating in Cuba (CRF19_cpx) was published online by the journal EBioMedicine. CRF stands for circulating recombinant form, and CRF19_cpx represents a combination of HIV-1 subtypes A, D and G. Last week, the authors issued a press release about the study, and since then media coverage has gradually ballooned. Most of the coverage takes its lead from the press release headline: “An aggressive form of HIV uncovered in Cuba.” The immediate problem is that the data are very preliminary, and it is way too soon to declare—as fact—that what has been uncovered is “an aggressive form of HIV.” It may or may not be, additional evidence is needed in order to make that determination. Many media stories also state that HIV CRF19_cpx causes AIDS within three years, which is potentially very misleading because what the study reports is that this rapid pace of progression was observed in a total of nine untreated people who were found to be infected with the strain; this does not prove that all people infected with HIV CRF19_cpx will progress at this rate. There is no evidence to suggest that HIV CRF19_cpx would not respond to treatment. 

The study was prompted by anecdotal reports from clinicians in Cuba that they are seeing more cases of rapid progression from HIV infection to AIDS. Starting in November 2007, a total of 95 untreated HIV-positive individuals who were “attending the Institute for Tropical Medicine ‘Pedro Kourí’ (IPK) for medical care” were recruited to participate in the study. A total of 52 were defined as rapid progressors based on a diagnosis of AIDS within three years of the estimated date of seroconversion (estimated as the mid-point between last seronegative HIV test and the first HIV-positive test). Criteria for an AIDS diagnosis were a CD4 count <200 cells, CD4 percentage <14% or the presence of an opportunistic infection. For the purposes of the study, rapid progressors were required to have met the CD4 T cell criteria for an AIDS diagnosis and have an opportunistic infection, or met the CD4 T cell criteria for an AIDS diagnosis and had at least two prior CD4 T cell counts <350. Of the remaining 43 participants, 21 had been followed for more than three years after seroconversion and had not progressed to AIDS (categorized as “non-AIDS”) while 22 had received an AIDS diagnosis three years or more after seroconversion (categorized as “chronic-AIDS”; these individuals had been followed since around 2001 and had retrospective data available).

Of the 52 cases of rapid disease progression, a total of nine were infected with HIV CRF19_cpx. The crux of the study is that this recombinant was not detected in any of the participants in the non-AIDS or chronic-AIDS groups, so it was overrepresented among rapid progressors. Using a Bayesian statistical model, the researchers demonstrate that infection with HIV CRF19_cpx was associated with rapid progression, as well as an increased likelihood of having oral thrush, higher levels of the chemokine RANTES, and greater prevalence of X4 tropism (as inferred from virus genotyping). There was also an association between HIV CRF19_cpx infection and having a higher viral load at diagnosis compared to other subtypes, but this analysis only involved six out of the nine participants. The fitness of HIV CRF19_cpx was estimated to be high, but based only on inference from genetic sequencing and not any assessments of virus replication.

The study results certainly call for additional investigation of disease progression rates in larger numbers of individuals infected with HIV CRF19_cpx. In the discussion section of the paper, several limitations are noted, including the absence of any information on the genetics of the study participants. The fact that 43 out of the 52 rapid progressors had other strains of HIV is consistent with the literature showing that non-viral factors such as immune responses and immune response genes play a key role in determining progression rates. These factors might have contributed to the outcomes in the individuals infected with HIV CRF19_cpx. One issue the paper does not address explicitly is why so many study participants progressed to AIDS without receiving treatment; in some cases, presumably, HIV infection and AIDS were diagnosed simultaneously, but it seems unlikely that this occurred in all the individuals (update: the senior author Anne-Mieke Vandamme has since explained via email that "all patients were treated according to the prevailing Cuban/WHO treatment guidelines" and the final version of the paper is being edited to include this information). The study offers absolutely no reason to believe that HIV CRF19_cpx wouldn't be suppressed by antiretroviral therapy just as effectively as any other HIV strain. 

Based on their press release, it appears the researchers wanted to highlight the relevance of their study to HIV prevention, as they begin by noting that acquisition of multiple strains of HIV can lead to the emergence of new recombinants. The release also expounds on a hypothesis offered in the paper that the apparently increased prevalence of X4 tropism in the nine HIV CRF19_cpx-infected participants was related to the high levels of RANTES and played a causative role in their rapid disease progression. The theory is that because RANTES binds to CCR5, the high levels seen in HIV CRF19_cpx infection favored the emergence of X4-tropism. However, although X4 tropism is associated with rapid CD4 T cell decline and disease progression, the cause and effect relationship remains uncertain. There are rare case reports of individuals genetically lacking CCR5 (CCR5Δ32 homozygotes) who have acquired infection with X4-tropic HIV strains, and while precipitous loss of CD4 T cells from the blood has been documented, this has not necessarily equated to the rapid onset of clinical disease. Also, there were concerns that CCR5-blocking drugs such as maraviroc might lead to rapid disease progression by selecting for X4 tropic HIV but this does not appear to have occurred in instances where X4 virus has emerged during treatment. Furthermore, no explanation is offered in the paper as to how HIV CRF19_cpx might cause increased production of the chemokine RANTES by immune cells.

The bottom line is that the paper raises the possibility that HIV CRF19_cpx is a particularly pathogenic HIV variant, but lacks the evidence necessary to confirm that this is the case.

EBioMedicine

doi:10.1016/j.ebiom.2015.01.015

Available online 28 January 2015

In Press, Accepted Manuscript

CRF19_cpx is an evolutionary fit HIV-1 variant strongly associated with rapid progression to AIDS in Cuba

Vivian Kouria, 1, Ricardo Khourib, c, , 1, Yoan Alemána, Yeissel Abrahantesa, Jurgen Vercauterenb, Andrea-Clemencia Pineda-Peñab, d, Kristof Theysb, Sarah Megensb, Michel Moutschene, Nico Pfeiferf, Johan Van Weyenberghb, Ana B. Péreza, Jorge Péreza, Lissette Péreza, Kristel Van Laethemb, Anne-Mieke Vandammeb, g

a Virology Department, Institute of Tropical Medicine Pedro Kourí, Autopista Novia del Mediodía Km 6, Marianao 13, Havana City, Cuba

b KULeuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Clinical and Epidemiological Virology, B-3000 Leuven, Belgium

c LIMI, Centro de Pesquisa Gonçalo Moniz, FIOCRUZ, Salvador-Bahia, Brasil

d Clinical and Molecular Infectious Diseases Group, Faculty of Sciences and Mathematics, Universidad del Rosario, Bogotá, Colombia

e AIDS Reference Center, Centre Hospitalier Universitaire de Liège, Liège, Belgium

f Department of Computational Biology and Applied Algorithmics, Max Planck Institute for Informatics, Campus E1 4, 66123 Saarbrücken, Germany

g Centro de Malária e outras Doenças Tropicais and Unidade de Microbiologia, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon, Portugal 

Received 17 October 2014, Revised 22 January 2015, Accepted 26 January 2015, Available online 28 January 2015 

Abstract

Background

Clinicians reported an increasing trend of rapid progression (RP) (AIDS within 3 years of infection) in Cuba.

Methods

Recently infected patients were prospectively sampled, 52 RP at AIDS diagnosis (AIDS-RP) and 21 without AIDS in the same time frame (non-AIDS). 22 patients were sampled at AIDS diagnosis (chronic-AIDS) retrospectively assessed as > 3 years infected. Clinical, demographic, virological, epidemiological and immunological data were collected. Pol and env sequences were used for subtyping, transmission cluster analysis, and prediction of resistance, co-receptor use and evolutionary fitness. Host, immunological and viral predictors of RP were explored through data mining.

Findings

Subtyping revealed 25 subtype B strains, 6 C, 7 CRF18_cpx, 9 CRF19_cpx, 29 BG-recombinants and other subtypes/URFs. All patients infected with CRF19 belonged to the AIDS-RP group. Data mining identified CRF19, oral candidiasis and RANTES levels as strongest predictors of AIDS-RP. CRF19 was more frequently predicted to use the CXCR4 co-receptor, had higher fitness scores in the protease region, and patients had higher viral load at diagnosis.

Interpretation

CRF19 is a recombinant of subtype D (C-part of Gag,PR, RT and nef), subtype A (N-part of Gag, Integrase, Env) and subtype G (Vif, Vpr, Vpu and C-part of Env). Since subtypes D and A have been associated with respectively faster and slower disease progression, our findings might indicate a fit PR driving high viral load, which in combination with co-infections may boost RANTES levels and thus CXCR4 use, potentially explaining the fast progression. We propose that CRF19 is evolutionary very fit and causing rapid progression to AIDS in many newly infected patients in Cuba.