Posts Tagged ‘therapy’

Plant-Based Antibodies, Vaccines and Biologics 5, Part 5

3 September, 2013

Session 6:Vaccines II

This was SUPPOSED to open with a report from Medicago Inc, on ‘Developing plant-made influenza vaccines: From discovery to commercial scale production’  – but didn’t, because they were all shaken up (in a good way) by having been effectively bought by Mitsubishi Tanabe Pharma Corporation, and no-one came.

This is a success story in its own right, however, as their recent and highly successful activities in the areas of making influenza vaccines and human rotavirus VLP-based vaccines in plants marked them out as a target for acquisition by Big(gish) Pharma – for which we commend them.

It is sad, however, that their only presence at the conference was on the back of my windbreaker B-)

Konstantin Musiychuk (Fraunhofer USA) was the first up, then, speaking on ‘Preclinical evaluation of VLP-based malaria transmission blocking vaccine’.  He described how there are 3 types of intervention that may work with malaria: these are at the pre-erythrocytic, blood stage, and transmission blocking stages of infection.  Antibodies to Pfs48/45, Pfs230 proteins block the fertility of or destroy the macrogamete.  Pfs25 and 28 Abs block the ookinete to oocyst developmental phase; all potentially block transmission.  Accordingly, they expressed these as fusions with the alfalfa mosaic virus (AMV) CP with mutation(s) to prevent glycosylation.  The Pfs25 protein was the best candidate; they cloned a mutated version (glyc-), fused at the N-terminus to AMV CP, and expressed via their TMV-based “launch vector” after vacuum infiltration.  He noted that the fusions have full-length and proteolysed products – which is needed for VLP formation as native CP is needed to avoid steric hindrance in assembly.  They obtained nice particles as shown by EM, showing surface decoration.  Dynamic light scattering [Ed: must get me one of those…] results show a nice tight range of 17nm particles.

They used the products with/out Alhydrogel as adjuvant, IM in mice: they got good titres maintained >170 days, with  2x inoculation.  They diluted test sera with naive human serum and used this to membrane-feed mosquitoes, then after 1 week dissected them and assayed for parasites: oocyte counts in mid-gut reflected efficient blocking of acquisition.  The adjuvant+ doses worked well down to 0.1 ug (100%).  Single doses of 1, 5 or 25 worked 100% as well.  After 6 months, 5 and 25 ug doses still gave 90%+ blocking.

They made GMP lots, very pure:  2 doses at 0 and 21 days resulted in complete blocking down to 0.3 ug, with >99% blocking after 40+ days.  Tox studies were fine, although the  Alhydrogel apparently causes some side effects.   Scaleup from 1-50 kg showed no changes in the Ag.  The Phase 1 trial is expected in Q3 2013.

This was most impressive: it is to be hoped that the promise is maintained!

Yoseph Shaaltiel (Protalix Biotherapeutics, Israel) spoke on Protalix’s new product: this was alpha galactosidase-A, for the treatment of Fabry disease.  This is an X-linked lysosomal storage disease that results in massive storage of glycolipid Gb3, in cells, in the vascular system and elswhere, which impairs the tissue of the heart and affects kidney and other organ function.  There were worse consequences than with Gaucher disease, while it was less obvious.  The current therapy was seen as being bad, and patients had reduced life expectancy.  There were 2 therapeutic enzymes on the market: these were Agalsidase Alfa and Beta; these were very inefficient and expensive, so cost benefit was very limited.  1/2 life in blood was normally just a few minutes, and the proteins were very immunogenic.

Protalix aimed at making a biobetter: this was made in tobacco cells cultured in bags (they used Icon vectors, so could not work in their favoured carrot), by cocultivation with Agrobacterium and then killing the bacteria.  The protein subunits were PEGylated to reduce immunogenicity and x-linked using bis-NHS-PEG.  This gave improved stability, longer circulatory 1/2 life, enhanced activity in target organs with similar to improved kinetics, so lower dosing and longer intervals between doses were possible.  Yields were good too, and they could make the enzyme very pure.  The product had the same kinetics as the commercial products with better activity over a wide pH range.

As far as glycosylation was concerned, the commercial product had very complex glycosylation, while the plant-made product’s profile was very consistent and simple.  It had an enhanced circulatory 1/2 life, of 581 vs 13 min, and also had higher activity in target cells – heart, kidney – over time.  Yoseph noted that the  patents on the enzyme(s) were limited to CHO cell production, meaning they had a useful window to exploit.

A comment from Jim Carrick was that the FDA was not interested in PEGylated products, as this could lead to vacuolation of kidneys in the long term.  Yoseph said their product was not the same, as normally PEGylation added 20-40 kDa, whereas theirs was a much shorter x-linker.  Their product was, moreover, already in clinics, as the  FDA had said they should move straight to patients rather than testing it in healthy people.

Lydia Meador (Arizona State University) reported on their lab’s HIV vaccine candidate, made in plants and also vectored by NYVAC-KC delB19 poxvirus.  They had previously shown that a CTB-HIV membrane proximal region (MPR) fusion vaccine resulted in Ab that stops transcytosis of HIV by Ab; she noted that live vectors enhance T-cell responses compared to subunit vaccines, so a combination would be a good idea.

Accordingly, they had cleverly produced whole HIV Gag and a deconstructed gp41 – stable Gag transgenics, and transiently-produced dgp41 – in the same plants, to make 100nm VLPs.  While VLPs are highly immunogenic alone, they wanted to prime with the NYVAC and boost with plant-made antigen.  They obtained good p24 Ab responses with NYVAC and the VLP boost; gp41 less so.  In terms of mucosal immunity, they saw the IgA response against gp41 was significantly higher in the NYVAC+VLP combination, as were CD8+ T-cells.  She noted that the anti-NYVAC titre was high after 3x doses.  In response to my question, she did not know if the NYVAC vaccine made VLPs in mice – which it may not do, even if it works in plants, due to different protein requirements for budding in mouse vs plant cells.

Daniel Tusé (Intrucept Biomedicine, Kentucky) – a company founded with Kenneth Palmer – spoke on ‘Safety and efficacy of plant-produced Griffithsin for antiviral indications’.  He noted that while griffithsin was an excellent anti-HIV microbicide, it was also a reasonably broad-spectrum antiviral lectin, as it was effective against the recently-emerged MERS CoV and  influenza viruses.

The protein was hard to make from seaweed, and E coli was useless for production; however, they got g/kg in tobacco via conventional rTMV vectors, and now even better with Icon and Nomad vectors.  KBP had manufactured it to near-GMP production standards, again at g/kg yields, with product recovery at 30% from leaves and 50% from leaves + stems, to a final purity of 99.8%.  The potency was the same as the alga-derived product, and they had 100s of gm of product.

As griffithsin binds HIV with very high affinity, its primary use would be as a topical microbicide, to prevent transmission of HIV and HSV; to prevent coronavirus infections, and to act on chronic virus infections.  The protein is not mitogenic on PBMC and does not activate T cells; it does not produce inflammatory cytokines in human PBMC, unlike cyanovirin, which had a much worse proinflammatory profile.  The epithelial toxicity was also very low, which was in contrast to some well-publicised agents which had disastrously resulted in increases of HIV acquisition in women using them.

A carbopol-based gel was found to have the best drug-release kinetics, so was adopted for formulating the product for use.  This protects mice against genital herpes: herpes has 2x the risk of infection per exposure compared to HIV infection.  The gel has broad specific activity against coronaviruses too, to a wide spectrum of viruses from human, cow, chicken and pig.  It could protect mice against SARS CoV, if given intranasally at 2 doses/day.

The protein also has uses in prevention of infection in the organ transplant area, eg against hepatitis C virus (HCV): it prevents infection of Huh-7 cells by cell-culture derived HCV, and partially protects hepatocytes from viral spread in vivo.  If injected in animals it persists, and maintains an anti-HIV activity.  It is immunogenic, but only weakly so, and Ab to it don’t neutralize its effects.  Their lab was using rational design to take out T-cell epitopes without affecting antiviral activity.

Daniel stressed that this is a new drug, which can be preferentially be made in plants at high yield, with very low cost of goods; that it was effective and safe.

Hugh Haydon (KBP) mentioned that the cost of goods was “pennies/dose”.

Session 8:

This was an interactive discussion session, addressing the topic ‘Commercialisation of molecular pharming products – objectives and targets for the next 5 years’.

The panel: from left - Hugh Haydon, Kevin Whaley, John Butler, Scott Deeter, Einat Brill

The panel: from left – Hugh Haydon, Kevin Whaley, John Butler, Scott Deeter, Einat Brill

Hugh Haydon of Kentucky BioProcessing (KBP), , speaking on behalf of the new MAPP, KBP and Icon collaboration, addressed product selection.  He noted that MAPP was responsible for product development, Icon for technology development and purification, and KBP for large-scale manufacture.  They had spun out Solmab as a collaborative vehicle for production of MAbs for infectious disease therapy.

He described their product selection rationale: this was based on

  • proof of concept data
  • platform suitability
  • capacity for dual use of product
  • availability of capital
  • speed of the regulatory process
  • regulatory success rate
  • scalability of existing infrastructure

Accordingly, they had selected a “biobetter” of Synagis, and an Ebola MAb cocktail.  The Synagis equivalent was better due platform parameters, known clinical parameters, the fact there were established markets which can grow, government and NGO humanitarian interest, and potential adaptation to other viruses.  For Ebola, they had a 3 MAb cocktail that was known to work, strong government interest (for a stockpile), a more rapid regulatory pathway, and a tropical disease voucher from the FDA.  He pointed out that these products won’t make blockbuster status, but are appropriate for small companies like theirs.

Kevin Whaley (MAPP) spoke on how we needed therapeutics that were multipurpose (disease, indication) as well as multi-vaccines.  The attributes of the new biologics were multi-use, speed of production, scale of production, and cost advantage – especially for global health products costing <$US10/g, at scales of >10K kgs, with increased efficacy (pathology, cancer), increased acceptability and access.  He noted that all modern paediatric vaccines are multi – this saves visits to clinics, especially in developing countries.

Scott Deeter (InVitria) noted that the biologics market was edging up to being worth $US125 billion – and reckons progress with plant-produced products is excellent.

John Butler (Bayer) thinks we are still looking for suitable products!  He was of the opinion that initial targets were too difficult (eg NHL – and flu??!), and that improved product characteristics must benefit from being plant-made.  He was adamant that PMP must not compete on price with other platforms – because there was no such thing as a bottleneck in fermentation capacity world-wide, and established industry could just cut prices if they wanted to.  He spoke of real and perceived hurdles:

  • regulatory pathway isn’t a hurdle
  • plant vs human glycosylation is not either, as plant-specific glycans were not more immunogenic than human

Real risks were that:

  • there were well-established alternatives
  • the plant-made product industry was overstretched in terms of resources

Einat Brill (Protalix) addressed their future strategy:

  • new biologics for orphan indications (clinical trials were smaller, one needed only several 10s kg a year for an entire disease cohort)
  • recombinant vaccines
  • hard to express proteins that were best expressed in plants

ApApproved biologics:

  • Biobetters of commercial products
  • They would continue to establish PMP regulatory environment as a viable route for biologic drugs development
  • Biobetter efficacy: longer circulatory half life for favourable clinical outcome
  • regimen frequency: longer treatment intervals due to increased drug stability, with lower dosing
  • Changing administration route (eg: oral vs injectable): helps to improve patient compliance

This was an excellent session, if only to hear how people who have been involved in getting PMPs to the market viewed the prospects for the industry – and it appeared favourable, despite John Butler’s caveats.

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Plant-Based Vaccines, Antibodies and Biologics 5: Part 1

27 June, 2013

Plant-Based Vaccines, Antibodies and Biologics: the 5th Conference

Verona, Italy, June 2013

The return of this biennial meeting to Verona – the third time it has been held here – was a welcome change; while the previous meeting in Porto in 2011 may have been good, the city was nothing like as pleasant a place to relax.  My group is now familiar enough with Verona that we know just where to go to get pasta by the riverside – or, on this occasion, “colt loin with braised onion and potatoes” and “stewed horse with red wine”.  Which seem more palatable, somehow, as “Costata di puledro con cipolle brasate e patate” and “Stracotto di cavallo speziata” respectively, but were enjoyed anyway.

The conference kicked off with an opening plenary session, chaired by the Local Organizing Chair, Mario Pezzotti, of the University of Verona.  The headline act was a talk on taliglucerase alfa – aka glucocerebrocidase, a Gaucher Disease therapeutic  –  by Einat Almon of Protalix Therapeutics from Carmiel, Israel.  I featured the product here last year, after an earlier feature here; suffice it to say that it has soared since FDA approval, and now Protalix is pushing hard with new plant-made products to follow it up.  While they use carrot cells for taliglucerase alfa, apparently they are using suspension-cultured tobacco cells for other products – and are using an easily-scalable disposable 800 litre plastic bag system, with air-driven mixing of cells suspended in very simple, completely mammal-derived product free media.  Hundreds of patients had been treated with the drug for up to 5 years with no ill effects, and the possibility of switching therapies from mammalian cell-made products to the plant cell-made had been successfully demonstrated.

Scott Deeter of Ventria Biosciences (Ft Collins, USA) spoke next, on “Commercializing plant-based therapeutics and bioreagents”.  His company has possibly the most pragmatic attitude to the production and sale of these substances that I have yet met, and he struck a number of chords with our thinking on the subject – which of course, post-dates theirs!  Ventria use self-pollinating transgenic cereals for production of seed containing the protein of interest, and rice in particular, for safety reasons – and because the processing of the seeds is very well understood, and the purification processes and schedules are common to many food products and so do not require new technology.  He reckoned that a company starting out in the business needed an approved product in order to give customers confidence – but should also engage in contract services and contract manufacture of client-driven products in order to avoid being a one-product shop.  To this end, they had received APHIS Biological Quality Manufacturing Systems (BQMS) certification (similar to ISO9001), with the help of the US Biotechnology Regulatory Services.

Their therapeutic products included diarrhoea, ulcerative colitis and osteoporosis therapeutics which were already in phase II clinical trial.  Scott noted that in particular, recombinant lactoferrin was a novel product, which could only feasibly be produced in the volumes and at the price required for effective therapy, by recombinant plant-based production systems.  It also filled a high unmet need as a therapy for antibiotic-associated diarrhoea in the US, with +/-3 million patients at risk annually who presently cost service providers over $1500 each for treatment.

A third commercialization option was bioreagents and industrial enzymes, which they marketed via a vehicle called InVitria: they had a number of products already in the market, which Scott claimed gave confidence to the market and to partners, while building capacity to make therapeutics.  Something that was particularly attractive to our prospects was that a collection or pool of small volume products – say $5-10 million each – gave a respectable portfolio.  He noted that Sigma Aldrich and Merck were already marketing their human serum albumin, which competed effectively with serum- and yeast-derived products.

George Lomonossoff from the John Innes Centre in Norwich, UK, spoke next on “Transient expression for the rapid production of virus-like particles in plants” – a subject close to our hearts, seeing as we have for the last five years been associated with George and partners in the Framework 7-funded PlaProVa consortium.  He mentioned as an object example the recent success in both production and an efficacy trial of complete Bluetongue virus (BTV) serotype 8 VLPs, made in Nicotiana benthamiana via transient expression using their proprietary Cowpea mosaic virus (CPMV) RNA2-derived pEAQ vector: this was published recently in Plant Biotechnology Journal.

Another very useful technology was the use of CPMV capsids as engineered nanoparticles: one can make empty VLPs of CPMV at high yield by co-expressing the coat protein (CP) precursor VP60 and the viral 24K protease: the particles are structurally very similar to virions in having a 0.85 nm pore at 5-fold rotational axes of symmetry, meaning they can be loaded with (for example) Co ions.  It is also possible to fuse targeting sequences – such as the familiar RGD loop – into the surface loops of the CPMV CPs, and to modify the inner surface too.  One application would be to engineer Cys residues exposed on the inside, which could bind Fe2+ ions: this would result in particles which could be targeted to cancer cells by specific sequences, then heated using magnetic fields.

John Butler of Bayer Innovation GmbH (Leverkusen, Germany) closed out the session with an account of lessons learned from the development of the plant-derived non-Hodgkins lymphoma (NHL) vaccine, that they had acquired with Icon Genetics, who in turn had inherited it from the sadly defunct Large Scale Biology Corp.  It was rather depressing to hear that Bayer had dumped the vaccine, despite the developers having reached their targets in turning 43 of 45 tumour samples into lifetime individualized supplies of vaccine within12 weeks, and despite the phase I trial being as successful as could be hoped.  To this end, the vaccines had been well tolerated and were immunogenic; of the patients who reacted immunologically, all but one were still tumour-free presently.

He felt that the problem was that NHL trials were too long and therefore too expensive as it was a slow-progressing disease; that a different clinical approach was needed, and that using the vaccines as a first-line therapy instead of only after the 2nd or 3rd relapse would be a much better idea.  The main lesson learned was that proving the technology would be far better done with a therapeutic vaccine for a fast-acting cancer, which would allow 1-2 year clinical trials with overall survival as an endpoint.

(more coming)

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Radical Therapy for HIV-Infected People?

18 May, 2012

To mark HIV Vaccine Awareness Day, 18th May – Journal Club – Lucian Duvenage:

Excision of HIV-1 Proviral DNA by Recombinant Cell Permeable Tre-Recombinase

Mariyanna, L., Priyadarshini, P., Hofmann-Sieber, H., Krepstakies, M., Walz, N., Grundhoff, A., Buchholz, F., Hildt, E., Hauber, J., 2012. Excision of HIV-1 proviral DNA by recombinant cell permeable tre-recombinase. PloS One 7, e31576.

Introduction

HIV Life Cycle. Russell Kightley Media, http://www.rkm.com.au

Highly active antiretroviral therapy (HAART) is a combination of drugs that has significantly elongated the lifespan HIV-infected people. HAART targets viral reverse transcriptase, protease and integrase. There are disadvantages including drug toxicity and the appearance of drug resistant HIV strains in people not adhering to or withdrawing from their treatment. There is a need for new therapies that not only block virus replication but also eliminate HIV from persistent viral reservoirs. An attractive option is Tre-recombinase, which been shown to excise provirus from the genomic DNA of infected cell cultures. The development of Tre recombinase is a previous publication (Sarkar, I., Hauber, I., Hauber, J., Buchholz, F., 2007. HIV-1 proviral DNA excision using an evolved recombinase. Science 316, 1912-5.)

The Tre recombinase was created from the Cre recombinase which is a well-known tool in mouse genetics. The authors were able to alter the specificity of the enzyme by many cycles of directed protein evolution.

The Cre recombinase precursor removes genomic DNA that is flanked by two loxP sites by recombination. The authors were able to alter the specificity for loxP sites to HIV-1 LTR (long terminal repeat) sites. LoxP and the HIV LTR had 50% sequence similarity.

The main problem with the development of antiviral agents is the delivery to infected cells in vivo, without causing adverse side effects.  There are many reported technologies for the delivery of macromolecules such as proteins, nucleic acids or peptides. The most popular currently is the use of protein transduction domains (PTD) also known as cell penetrating peptides (CPP) from different sources. These have been useful for the delivery of various genes and proteins, including site-specific recombinases. The best studied and most applied PTD’s are peptides derived from the basic domain of HIV-1 Tat. But recently a powerful cell permeable translocation motif (TLM) has been described. This TLM is derived from a hepatitis B virus surface antigen. This TLM peptide is able to enter cells very efficiently, without affecting the integrity of the cells, or interfering with intracellular signal transduction cascades.

This paper describes the delivery of Tre-recombinase into cells using these PTD’s including HIV Tat and the HBV TLM. These so-called cell-permeable Tre-recombinases could eventually be useful for antiretroviral therapy, especially for virus eradication.

Results

Proteins

Different protein fusions were created and expressed in E. coli ; Tre-recombinase is fused to His tag, with/without nuclear localisation signal (NLS) and with the PTD (HIV Tat) or TLM (translocation motif derived from hepatitis B) or TLM as an inverted repeat.

They tested the cellular toxicity of the protein at their highest concentration by alarmBlue assay in HeLa cells. The proteins were incubated with the cells for 48 h. None of the proteins had any significant effect on the cellular metabolism

Cell permeability

Interestingly, all of the proteins entered cells, even those without a PTD or nuclear translocation signal. The authors explain that the Cre enzyme precursor to Tre has been shown to transduce into mammalian cells without any help, and therefore it is likely that the Tre enzyme shares this property. The authors did remark that the signal intensities were higher for those proteins with a PTD, indicating higher transduction efficiency.

Analysis of Tre activity in HeLa cells

A transient reporter assay demonstrated the activity of the Tre fusions: The reporter construct contains the target LTR sites that flank a puromycin resistance gene. Tre enzyme activity results in the loss of this gene, and gives a smaller PCR product using primers that anneal to the vector backbone. Cells transfected with the reporter construct were incubated with the 1 µM of the various proteins for 5 hours. The positive control was co-transfected with a construct expressing the Tre enzyme. PCR was performed on DNA extracted from cells after 48 hours. The presence of the smaller PCR product indicated that recombination had happened, as in the positive control. All of the proteins had varying degrees of activity, but notably the protein with the TLM PTD had the highest activity, with no un-recombined product detected by the PCR.

The authors went on to demonstrate that the Tre fusion proteins were active on at the genomic level, i.e. on chromosomal DNA. They used cells with the reporter construct was stably integrated into the genome.

Interaction of proteins with LTR sequences in living T-cells

Co-immunoprecipitation (ChIP) assays were done on using HIV-1 -infected T-cells (CEM-SS) to demonstrate the interaction of two of the Tre fusion proteins with the HIV LTR target sequences. The results showed that the proteins interacted with target LTR sequences in the genome of infected T-cells.

Microarray

The authors performed a transcriptome anaylsis on cells exposed to the Tre fusion proteins, using human whole genome microarrays. They concluded that the proteins were unlikely to have a significant effect on gene expression in the host cells, as very few genes were regulated more than 2.5-fold.

Recombination of the full-length HIV proviral genome:

Up to this point, the Tre fusion proteins had been shown to be capable of excising reporter construct gene flanked by LTR sequences both at the episomal level and the chromosomal level. The authors also showed that the proteins bind to the target sequences in HIV-infected living T cells.

It was essential that the Tre fusions could excise that HIV proviral genome from the chromosomal DNA of HIV-infected cells. The aouthors generated HeLa cells and T cells infected with pseudotype HIV-1. These are cells with the full-length HIV provirus integrated into the genome.They chose one of their proteins (TLM fusion showing highest activity in the reporter assays) for transduction into these cells. After transduction, PCR was performed to detect the HIV circular recombination product. They found that the recombination activity increased in a dose-dependent manner in both the HeLA cells and the T-cells. They also sequenced the PC products and were able to confirm HIV sequences.

Discussion

Some novel therapies for the treatment of HIV focus on the eradication of the virus in infected individuals. These include RNA-based technologies such as RNA aptamers, siRNA and ribozymes, but while these have shown to reduce viral load and viral replication, they have so far failed in virus eradication. A recent approach aimed at virus eradication is the reduction of surface CCR-5 receptors, through the expression of engineered zinc finger nucleases. This results in fewer CCR-5 surface receptors and could prevent new infection by CCR-5 tropic HIV.

The other approach is the use of site specific recombinases like Tre, which can excise the provirus from the host genome, thus potentially eradicating the virus from the individual. Ideally, the gene expressing Tre could be delivered to and expressed in target cells using a viral vector. But there are safety concerns as most of these are derived from pathogenic viruses. Therefore it may be advantageous to deliver the Tre enzyme directly to host cells. One way of doing this is through protein translocation domains (PTD’s). Protein transduction domains (PTD’s) can deliver bioactive molecules, including genes, siRNA, proteins or liposomes into all types of cells in vitro and furthermore into various organs in vivo. But they have not been applied yet for human use. PTD’s are easily fused to any target protein through cloning and expression of the fusion protein. The LTM used in this paper, derived from hepatitis B virus surface antigen, has low immunogenicity and high spreading capacity.

One strategy of using cell-permeable Tre enyme could be to harvest T-cells from the patient by apheresis and transduce them in vitro. They could then potentially be expanded and reinfused into the patient. This could complement or even replace gene transfer procedures.

In this paper the expressed Tre fusion proteins could enter cells and act on the target sequences to excise the HIV provirus from the genome, but the Tat fusion and the TLM fusion had higher activity than those that didn’t have a PTD tag. In particular, fusion to the newly described domain (TLM) from hepatitis B was resulted highest activity. This may explained by the fact that, in contrast to HIV Tat, TLM does not rely on endocytosis to enter cells. This might preserve enzyme activity and protein half-life.

In conclusion, cell permeable Tre enzyme could eventually be useful as an anti-HIV therapy in the post-HAART era.

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Posted in General Virology, HIV, Vaccines: General | 2 Comments »