Faculty of Medicine, Dentistry and Health Sciences Department of Microbiology and Immunology

Antibody dependent cellular cytotoxicity - a neglected anti-HIV immune response!

CD56+ CD3- Natural Killer
lymphocytes expressing
IFNϒ in response to HIV+
serum and ADCC peptide
epitope within Vpu.
No IFNg is expressed to no
peptide, an irrelevant peptide,
or the absence of IgG.

The importance of HIV-specific Antibody-dependent Cellular Cytotoxicity (ADCC) antibodies that inhibit viral replication mediated through the Fc receptor is becoming clear. In view of the difficulty of inducing broad neutralizing antibodies and effective T cell immunity, exploring HIV-specific ADCC is a priority. HIV-specific ADCC has been under-explored both from the point of view of rapid, reliable assays to measure and characterize ADCC responses and the ability to specifically induce high level HIV-specific ADCC antibodies by vaccination. Recent data from the RV144 vaccine study in Thailand suggest a role for non-neutralising antibodies in protection from HIV.

We recently identified a simple flow cytometry technique to identify HIV-specific ADCC responses and map ADCC epitopes. In its simplest form, small volumes (200ul) of whole blood from HIV-infected subjects are stimulated for 6hr with Env protein or overlapping Env 15mer peptides (or whole Env proteins), and CD56+ NK lymphocytes assessed for expressing the cytokine IFN-y and/or the degranulation marker CD107a. The activity is mediated by IgG antibodies in plasma and the assay can be performed on stored plasma samples using donor blood or PBMC as effector NK cells. The ADCC epitopes can readily be finely mapped to individual peptides from within an overlapping set. The ability of the HIV-specific ADCC antibodies to trigger "polyfunctional" NK cells (e.g. express TNF-alpha, degranulated perforin or granzymes, express chemokines) is also readily monitored in this assay since the NK cells can be accurately gated on. We recently showed ADCC antibodies trigger very rapid activation of NK cells (Chung et al, Journal of Immunology, 2009), which could be important in limiting virus spread. We are now endeavoring to isolate specific ADCC antibodies and identify the most effective antibodies to use in passive transfer studies. We recently wrote a lay review of this area.

Exciting new data shows that HIV-specific ADCC responses can force immune escape. This strongly suggests ADCC applies significant pressure to HIV. This was published in PNAS and attracted some media interest here. Reports appeared in The Age and Science Illustrated amongst others. We also recent found that people with HIV that progress more slowly to AIDS have enhanced ADCC responses to specific parts of HIV (see here). Dr Matt Parsons joined our group to study the role of NK cells in the ADCC process (see here). Dr Marit Kramski is now investigating how ADCC might help clear cell with latent HIV and thus help in the search for a HIV cure - we recently wrote a review in this area (see here).

Personnel: Dr Ivan Stratov, Dr Marit Kramski, Dr Matt Parsons, Ms Vijaya Madhavi

Funding: NHMRC Project Grant #1034770, Clive and Vera Ramaciotti Foundation for Medical Research Award, Royal Australasian College of Physicians CSL Medical Research Scholarship, Australian Centre for HIV and Hepatitis Virology

Combined Influenza-AIDS Vaccines

FACS plots of SIV KP9-specific CD8+ T cells in blood
before and 7 days after Flu-SIV infection

Influenza and HIV are both serious global pathogens. Can a single vaccine be designed to cover both viruses?! Recent advances in reverse genetic techniques allow insertion of foreign antigens into live influenza viruses. Further, live attenuated influenza vaccines are now highly effective vaccines. Together with collaborators Lori Brown, Brad Gilbertson, John Stambas, Steve Turner and Nobel laureate Peter Doherty we are designing recombinant influenza vaccines with inserted SIV antigens to test as a combined Influenza-AIDS vaccine. An advantage of this strategy is that as a mucosal virus, there is a strong likelihood that immunity at mucosal surfaces, where HIV is first encountered, can be induced with this approach. Exciting preliminary data suggest this approach has great potential to induce T cell immunity although escape from single CTL epitopes is a significant barrier. We are now generating Flu-HIV vaccines that express an expanded range of HIV antigens..

Personnel: Dr Rob De Rose, Mr Hyon-Xhi-Tan, Ms Sheilajen Alcantara.

Funding: NHMRC Project Grant #628331, Gates Grand Challenges Exploration Grant #OPP1008294.

Nanoparticle HIV vaccines

No safe HIV vaccines have been able to stimulate durable, activated T-cell immunity. We are investigating an exciting HIV vaccination approach using hollow, submicron, delivery vehicles (nanocapsules) assembled using layer-by-layer technology. This is a novel, cross-disciplinary project with the Caruso group at University of Melbourne. Nanocapsules are designed to induce optimal immune responses by protecting antigens from degradation prior to reaching sites of immune activation and activate antigen presenting cells in a way that will initiate anti-viral immune responses. We aim to use controlled-release nanocapsules to stimulate durable CD4 and CD8 T cell responses in vivo.

Our work to date (De Rose et al., Advanced Materials, 2008; Sexton et al., ACS Nano, 2009; Chong et al., Biomaterials, 2009) has shown that LbL nanoparticles are efficiently endocytosed by dendritic cells and monocytes in fresh whole blood in a dose dependent manner. Furthermore, we have assembled LbL nanocapsules containing an SIV Gag peptide KP9 and demonstrated that when KP9-nanocapsules are incubated with blood from a Mane-A*10 positive macaque, KP9-specific T cells are stimulated to secrete cytokines (De Rose et al, Advanced Materials, 2008).

These exciting initial findings have been achieved with standard nanoparticles prepared from a range of different polymers that are simple in design and can be prepared rapidly. We are now testing these nanocapsules HIV vaccines in animal models. Further, we are utilising the enormous flexibility of this approach to incorporate novel adjuvants to improve the immunogenicity of these vaccines. In addition, we are also investigating the use of these particles to target anti-HIV inhibitory RNAs to HIV-infected CD4 T cells as a therapeutic strategy. As part of this strategy we have been invesigating novel ways to avoid non-specific uptake of particles - so called "stealth" particles.

<Personnel: Dr Rob De Rose,Prof Stephen Kent, Ms Sheilajen Alcantara, Mr Hyon-Xhi Tan in collaboration with Prof Frank Caruso, Dr Jiwei Cui and Dr Angus Johnston.

Funding: Strategic Research Initiative Funding, University of Melbourn;, Australian Centre for HIV and Hepatitis Virology Research (ACH2) Award.

NKT cells, MAIT cells and HIV

NKT cell population in Macaca
Nemestrina blood

NKT cells are a small but very important lymphocyte population in blood that possess potent antiviral and antitumor activities. In collaboration with Dr Godfrey and colleagues, we recently showed NKT cells are depleted during SIV and SHIV infection of monkeys (Fernandez et al, Journal of Virology, 2009). We are now devising strategies to enhance the biologic utility of NKT cells in fighting HIV. We are also starting to study an interesting population of cells called Mucosal-associated Invariant T cells (MAIT cells).

Personnel: Dr Stephen Kent, Prof Dale Godfrey, Ms Caroline Fernandez

Funding: NHMRC Program Grant #510448

Checkmating HIV - trapping immune escape HIV strains

CD8+ cytotoxic T lymphocytes (CTL) can be effective at controlling HIV-1 in humans and SIV in macaques, but their utility is partly offset by mutational escape. The kinetics of CTL escape, and reversion of escape mutant viruses upon transmission to MHC-mismatched hosts can help understand CTL-mediated viral control and the fitness cost extracted by immune escape mutation. Traditional methods to follow CTL escape and reversion cannot, however, detect minor viral quasispecies. We recently developed sensitive quantitative real-time PCR assays to track the viral load of wild-type and escape mutant SIV variants in pigtail macaques. Rapid outgrowth of escape mutant virus occurs during the first few weeks on infection. However, the rate of escape plateaus soon after, revealing a prolonged persistence of wild type virus in blood. The rate of reversion to wild type slows dramatically after the first month of infection. Both vaccination ( Loh et al, PloS Pathogens, 2008) and the make-up of the infecting virus (Loh et al, PloS Pathogens, 2009) affect the rate of escape and reversion. This work has implications for how best to control HIV during early infection.

We are now applying similar techniques to the study of the integrated virus reservoir. Formation of integrated virus is a key blockade to curing HIV. The precise timing of when reservoir viruses are laid down has however been difficult to study in humans since the infecting isolate is usually not known and serial samples are rarely available during the first 1-2 weeks of acute infection prior to symptoms. This work was recently highlighted in The University of Melbourne Voice (Vol.5, No.2 May 2009)..

Together with our collaborators Drs Miles Davenport and Janka Petravic at UNSW, we are defining when and how much wild-type and escape mutant virus is replicating during infection, this effectively provides a “molecular clock” to track when the We find that there is rapid turnover of viral DNA in resting CD4 T cells during active replication with high viral loads - this has implications for new strategies to reduce or eliminate the latent reservoir and approaches to cure HIV. This work was recently published in Plos Pathogens. We are now evaluating escape variants in the latent reservoir when antiretroviral therapy is initiated.

Personnel: Dr Stephen Kent, Dr Jeanette Reece, Shayarana Gooneratne, Thakshila Amaresena

Funding: NHMRC Project Grant #454553, AmfAR Award #106849-42-RGRL, ARC Discovery Project #DP0987339

DC targetting vaccines

Generating robust immune responses requires delivering vaccines to Dendritic cells to initiate immune responses. We are collaborating with Drs Mireille Lahoud, Irene Caminschi, Andrew Lew, and Ken Shortman from WEHI and the Burnet who have developed clever antibodies that target a molecule on DCs called Clec9A. These antibodies can be linked to vaccine antigens
to promote high level immunity. This has worked very well in mice and we are now further developing this concept.

Personnel: Dr Stephen Kent, Dr Rob De Rose, Sheilajen Alcantara

Funding: WEHI Catalyst award