Posts Tagged ‘HIV’

Emergency response vaccines for H5N1 influenza in South Africa

1 November, 2013

Our group has been working for some time now – since 2006, in fact – on investigating the feasibility of providing South (and southern) Africa with emergency response pandemic influenza vaccines.  The research was initiated after the Virology Africa 2005 conference that Anna-Lise Williamson and I organised in the Cape Town Waterfront in November of that year – when a senior WHO official warned us in his talk that “…if a pandemic hits, you are on your own: no-one will give you any vaccine”.

A group of us sat down afterwards, and discussed the feasibility of looking at emergency response vaccine(s), given that we had no capability in the whole of Africa to make flu vaccines.  Anna-Lise and I put together a proposal, with the highly pathogenic avian H5N1 influenza A as a target, which was funded on a once-off one-year basis by the Poliomyelitis Research Foundation (PRF) here in SA for 2006 – and then again by the PRF as a three-year Major Impact Project  (MIP) from 2008-2010, and subsequently to a lower level by both the PRF and the Medical Research Council of SA.  What made it all the more impressive for a South African project was that we had proposed expressing a protein-based vaccine in plants – quite a revolutionary prospect at the time, but something that followed on from the highly successful production of Human papillomavirus virus-like particles by transient expression in Nicotiana benthamiana by  James Maclean, working as a postdoc in our lab at the time.

However, some of the most important work was done early: James was very quick to get the haemagglutinin (HA) gene for the A/Vietnam/1194/2004 strain of H5N1 synthesised by GeneArt in Germany, and cloned into the same Agrobacterium tumefaciens plant expression vectors from Professor Rainer Fischer’s lab in Aachen, Germany, that had been used for HPV.  His initial work showed that large amounts of HA protein could be produced, both as soluble protein which lacked a membrane localisation domain, and as the membrane-bound form.  This work formed the basis for a patent application on the transient expression of H5 HA that has now been granted.

Subsequently, when the PRF MIP started, we employed Dr Elizabeth (Liezl) Mortimer and Ms Sandiswa Mbewana to further the work: with collaborators from the National Institute for Communicable Diseases (NICD) in Johannesburg and State Veterinary Services in Stellenbosch, this investigated transient and transgenic expression of soluble and membrane-bound forms and their immunogenicity, as well as a DNA vaccine consisting of the HA genes cloned into Tomas Hanke’s pTH vector.

The protein expression work was published in 2012, as well as being featured here in ViroBlogy at the time.

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What we had managed to show was that we could get excellent production of the H5 HA in both soluble and bound forms, and that especially the membrane-associated form of the protein was highly immunogenic, and elicited antibodies in experimental animals that were appropriately neutralising, indicating its suitability as a vaccine candidate.

Now this all happened despite our running out of money AND Liezl leaving to have a baby…and then we managed to get another paper out of the work, this time on the DNA vaccine side of things.

Image

We pitched this at the South African Journal of Science as a vindication of the faith in us by exclusively South African funding agencies – and managed to get the cover of the issue in which it appears, thanks to the truly excellent artwork of Russell Kightley from Canberra, Australia.  Front AND back covers, as it happens…!

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And this all made Sandiswa Mbewana, who is now a PhD student on another project, very happy:

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This all came in excellent time to mark the establishment in the Department of Molecular and Cell Biology at the University of Cape Town, of a new URC Research Unit: namely, the Biopharming Research Unit (BRU).

BRU

Watch this space…B-)

Plant-Based Vaccines, Antibodies and Biologics 5, Part 4

2 September, 2013

PBVAB 5 Part 4

Sessions 5 – 8

The fifth session on Day 2 was “Antibodies 1” – and who better to kick off, than Rainer Fischer (RWTH / Fraunhofer Institute, Aachen), talking about Pharma-Planta – The European project to introduce plant-derived monoclonal antibodies to the clinic’.

One of the most impressive features of the FP6 PharmaPlanta project was its sheer size: 28 academic institutions were involved over 7 years, at a cost of €12 million plus €3 million from the Fraunhofer Institute in Aachen.  Their mission was to move molecular farming beyond proofs of concept, and to develop candidate products.  They selected the anti-HIV-1 subtype B MAb 2G12 as their final candidate, but also developed MAbs to rabies and some vaccine candidates.  Importantly, their IP had a Humanitarian Use Commitment: knowledge created was made freely available for humanitarian purposes.

They had a total of 39 postdocs and 8 students trained; they produced 200 peer-reviewed publications consisting of 150 research papers and 50 reviews, and a spin-out company.  The project also helped to develop a South African plant-made MAb production platform.  Their plant-produced 2G12 was the first plant-made MAb in human clinical trials – and went from gene to clinic in just 7 years.  They had also very materially helped the development of the regulatory regime in Europe, from the viewpoint of pharmaceutical guidelines and environmental safety for PMPs.

Rainer Fischer

Rainer Fischer in full flow

The final yield figures for 2G12 were 5 g of 97% pure MAb from 240 kg of transgenic tobacco, with a recovery of 55%.  The product had a better glycosylation homogeneity than CHO cell-produced 2G12.  In clinical trials of the MAb used as a vaginal microbicide, the product was safe and well tolerated with no serious adverse reactions.  There were no anti-Abs found in serum or in the vagina, with no systemic absorption.  The MAb survived for 8 hrs in the vagina, meaning it had serious potential as a microcode.

The project resulted in great human capital, a manufacturing facility at the Fraunhofer IME, and a number of important follow-on projects.  It also opened bottlenecks in regulatory practice, and in clinical trials of PMPs.  There was a pipeline of additional product candidates, eg anti-rabies MAbs.

Important lessons from the project were the following: one should focus early on on the plants used, the expression technology, the threshold level of production, realistic timelines, the plant line and purification process, production issues, QC stability, regulatory contract – FIND A CLINICAL SPONSOR!, set up contractual framework, draft specifications for drugs, contact authorities in countries for manufacture and testing.

Issues such as smart product selection, synthetic biology/host cell line engineering, glycan/protease profile, hi-throughput cloning, selection of elite lines, scale-up automation / vertical farming, downstream processing, regulatory approval had also surfaced, and were important.

For the future, a fully automated vertical farm unit  for seed development was going to come on stream.  They would move from niche production to mainstream production, taking advantage of economies of scale.  Other developments could be designing an optimal host cell line, with fully human glycosylation, and site-directed transgene integration.

Some day someone should write a book about this endeavour – and I think it should be Rainer.

Larry Zeitlin (MAPP Biopharmaceutical) spoke next, on producing monoclonals against respiratory syncytial virus (RSV): the reason for doing this is that RSV is a major pathogen among small children worldwide, and while there are MAb-based therapeutics (eg: Synagis, from MedImmune), with sales in the order of USD 1 billion annually, these cost around USD 5 000 for one treatment for one child – and premature infants or cardiac / respiratorily challenged children required 4-5 monthly doses per RSV season.  Additionally, infection with RSV in the 1st year of life is associated with development of asthma later, so paediatricians were wanting to treat a much wider spectrum of children.

Accordingly, MAPP was making a Synagis equivalent via Icon vectors in N benthamiana for half the cost of goods, which had the same neutralisation ability and same affinity but a different glycosylation profile and shorter half-life.  When tested in cotton rats it was identical in pharmacokinetics and worked as well as Synagis.  An attempt to reduce the interaction of the IgG1-based MAb with the immune system by changing the subtype to IgG2 failed in rates even though it was neutralising, possibly due to there being less ADCC.  Larry mentioned that they could engineer the Fc region with point mutations to significantly extend the half life – and then use this as a scaffold, possibly for some of their other products.

Michael McLean (Univ Guelph, Canada) described his group’s work on a HIV Ab cocktail theoretically capable of neutralising 99% of HIV strains – this was for PlantForm Corp, who had a mandate to produce biosimilars and novel biologics using plants.  The HIV project was focused presently on demonstrating anti-HIV functionality, and at improving glycosylation profiles of a cocktail of b12, 2F5 and 4E10 broad-spectrum anti-HIV MAbs.

They worked with BeYDV-derived, 2-replicon vectors expressing whole MAbs, as well as their own vectors, using the Steinkellner group glycosylation pathway engineered plants.  With 9 days maximum expression period  they could get 1 g/kg maximum yields.  All the MAbs worked fine, with  similar activity in in vitro HIV pseudovirion neutralisation assays.  Using the deltaFX N benth line, they get uniform glycosylation – and add Gal using their own vectors.

Shawn Chen (BioDesign Inst, Arizona State Univ) described their work on a humanized West Nile virus (WNV) therapeutic MAb which protected mice from WNV infection.  They wanted blood-brain barrier (BBB)-permeable bifunctional Abs to extend efficacy, presently limited because of the barrier.  They got 0.3 – 0.5 g/kg yield of a bifunctional MAb which bound the BBB endothelial receptor and virus Ag, using Icon and BeYDV vectors, and showed endocytosis into brain cells.  He also mentioned that they could “tune” glycoforms to change ADCC.

IMG_0140

Victor Klimyuk (Icon Genetics GmbH, Germany) presented on ‘Biogeneric antibodies made in plants’: these used a generic IgG1 constant region gene codon-optimised for plants, with add-on variable (V) regions derived from other Abs of different types and specificities.  The first product had been the non-Hodgkin lymphoma personalized MAbs: they had done glycotyping of each NHL MAb, all with the same H but diff L chains, to show these were differently glycosylated – and that all the idiotypes were expressed at very different levels.  Interestingly, expression levels had little to do with occupancy of glycosylation sites – and this occupancy could be tuned by directed point mutations.

They had made analogues of trastuzumab and herceptin, etc – and noted that herceptin analogues differed in potency, and wt plants produced lower levels than their engineered plants.  Rituximab analogues were all the same as the original MAb at day 0 of treatment, but MAbs with no fucose were best at persistence – equal to the original.

Vikram Virdi (VIB, University of Gent, Belgium) described passive immunisation of piglets against enterotoxigenic E coli (ETEC) using llama-derived antibodies produced in Arabidopsis.  This was useful in that it extended the maternally-derived passive immunity.  Their product was a “porcinised camellid Ab” against the major adhesion molecule of ETEC, which should survive the digestive tract.  They made MAbs based on a camellid Vh gene fused to IgG and IgA Fc regions, and expressed them in seeds for a piglet feed challenge.  They got a maximum of 15% TSP expressed in seed, 3% of seed weight.  By triple transformation with the 3 genes required for an IgA analogue (Vh:Fc, J chain and secretory component) and then selfing and breeding plant lines, they got in planta assembly of a sIgA analogue (0.2% seed weight).  This worked in inhibiting attachment of  bacteria, so they upscaled production and tested a cocktail of IgG vs IgA types.  The latter was best, with a swift decline of bacterial shedding with a 4  x lower dose than for IgG.  There was also a better weight gain for IgA treated piglets.

Thomas de Meyer (VIB-PSB/University of Gent) compared production of bivalent camellid VHH-derived MAbs in Arabidopsis, N benthamiana and Pichia pastoris, given that the VHH Fc enhanced functional affinity, and led to longer serum 1/2 life, and was a convenient protein tag. They compared VHH and VHH-Fc MAbs with 4 fusions, including anti-globulin, anti-albumin, and anti-GFP.  The products were stable in seed production (with KDEL) in Arabidopsis and also N benthamiana, and  Pichia secreted the products.  They got yields of 1.5 – 27% TSP, 0.1 to 0.82 g/kg in plants, and with Pichia, 15 – 30 mg/l culture.

The MAbs had different size profiles from the different hosts, though all were bivalent VHH, and N benthamiana and Pichia products were fully glycosylated.  Several of the Fc-type MAbs outperformed the VHHs in ELISA.

Overall, it was obvious that expression of a wide variety of antibodies in plants is a maturing technology: yields are high, of antibodies whose glycosylation and retention profiles can be handily engineered, and which perorm equivalently or better than their conventional homologues in in vitro and in vivo assays.

Go Green, he said, not quietly…B-)

ViroBlogy: 2012 in review

1 February, 2013

So: thank you, anyone who clicked in, and regular visitors.  You make it worthwhile!!

The WordPress.com stats helper monkeys prepared a 2012 annual report for this blog.

Here’s an excerpt:

4,329 films were submitted to the 2012 Cannes Film Festival. This blog had 33,000 views in 2012. If each view were a film, this blog would power 8 Film Festivals

Click here to see the complete report.

Virus-like particle and Nano-particle vaccines 2012: a conference report

30 January, 2013

Alta van Zyl, Virology Group, Molecular & Cell Biology Department, UCT

Introduction:

VLP flusm

Haemagglutinin-only Influenza A virus VLP. Courtesy of Russell Kightley Media

The new international conference on virus-like particles and nano-particles (VLPNPV) took place in Cannes, France at The Novotel Montfleury Hotel from the 28th to the 30th of November 2012.  The scope of the conference included virus-like particles (VLPs), the plant-based expression of VLP vaccines as well as expression and optimisation of VLPs.

Other topics included in the conference were:

  • VLP platform delivery systems
  • VLP vaccines
  • Nano-particles and nano-particulate vaccines

A multitude of topics were covered during the conference and many of the talks pertained to the immunogenicity of the VLPs and nano-particles and how they compared with the immunogenicity of DNA or subunit vaccines.

Talks were given by researchers from companies such as Medicago, Mucosis, Pevion Vaccines and Novavax. These talks gave a perspective on factors that need to be considered when commercialising VLP/nano-particle vaccines and to be GMP compliant.

Compelling presentations:

Developing plant-made virus-like particle vaccine products: An integrated platform from discovery to commercial scale

Marc-Andre D’Aoust, Nathalie Landry, Sonia Trepanier, Michele Dargis, Manon Couture and Louis-Philippe Vezina (Medicago, Quebec City, Quebec, Canada)

This talk was about a plant-made VLP against both pandemic and seasonal influenza- these vaccines are now in the clinical trial phase. What was especially interesting was the view from an industry point of view where expression had to be scaled up to produce large amounts of vaccine.  The Medicago platform can synthesize and clone approximately 100 gene constructs in two weeks, they can prepare 100 bacterial cultures per week and they have automated infiltration where 200 plant transformations can be performed per day and 150 VLP engineering approaches can be tested in one week.  For influenza Medicago tested 48 different infiltration approaches in one day for HA, NA, M1, M2 as well as P1 Gag and HGalT.  Medicago has been able to produce 10 million doses of HA VLPs in just one month.

See also: 

  • D’Aoust et al (2010) PBJ 8:  607-619 – The production of hemagglutinin-based virus-like particles in plants: a rapid, efficient and safe response to pandemic influenza.
  • http://www.medicago.com

Development of RNA-free plant VLPs a source of novel therapeutics

George Lomonossoff (John Innes Centre, Norwich, UK)

This group made empty Cowpea Mosaic Virus (CPMV) VLPs that contained no RNA.  CPMV VLPs are versatile nanoparticles to which organic, inorganic and biological molecules can be bound.  The empty nature of the particle means that they can be used as carrier molecules for therapies; this could prove to be potentially useful as a cancer-treatment therapy.  The system is advantageous because of the lack of RNA which makes the particles non-infectious and no bio-containment is needed for the production of these VLPs.

Immunogenicity of VLPs: an immunological perspective

Martin Bachmann (University of Zurich, Zurich, Switzerland)

Background was given from immunological point of view about what makes VLPs so immunogenic. Three properties contribute to the immunological properties of VLPs (1) their size, (2) the repetitiveness of the particle capsid which provides multiple sites for antibody binding and (3) TLR ligands – the particle can be disassembled, the RNA removed and replaced with a TLR ligand to enhance immunogenicity. Also, the size of VLPs is optimal for drainage to the lymph nodes.

Immunogenicity optimization strategies for public-sector development of vaccines: the critical role of optimizing the antigen.

Martin Howell Friede (WHO, Geneva, Switzerland)

This talk was about looking at VLPs from the vaccine development view.  Monomeric antigens are not very immunogenic; therefore adjuvants were developed and came into use. For an efficient vaccine the antigen must be multimeric as antigen alone is insufficient to be immunogenic without adjuvant. Two factors have to be considered when producing a vaccine for FDA approval; (1) optimise the antigen before using an adjuvant, (2) use an adjuvant that has already been approved by the FDA. VLPs as vaccines provide the potential for immune-stimulation without the addition of adjuvant as the multimeric presentation of the antigen will enhance its immunogenicity.

Enhancing the immunogenicity of VLP vaccines

Richard W. Compans (Emory University, Atlanta, Georgia, USA)

This talk highlighted strategies which could be used to enhance the immunogenicity of VLPs.

  1. Look at alternate routes for vaccine delivery (intranasal, intramuscular, subcutaneous etc)
  2. Increase the breadth of immunity by enhancing responses to conserved antigens/epitopes
  3. Increase the amount of antigen incorporated into VLPs
  4. Incorporate the adjuvant into the VLPs as part of the structure

See also:

  • Ye et al (2011) PLoS One 6(5):  e14813
  • Wang et al (2008) J Virol

Innate and adaptive responses to plant-made VLP vaccines

Brian Ward (McGill University, Montreal, Quebec, Canada)

Brain Ward is also the medical officer at Medicago.  Humans rarely react to plant proteins/antigens. The plant glycans fucose/xylose at the N-terminal is an allergen and can cause anaphylaxis in humans. During trial experiments with influenza no individuals developed IgE responses to plant glycans, therefore plant produced vaccine is safe. The H1 VLP induced long lasting memory multifunctional T-cell responses in humans.

Impressions of the conference:

The conference was well organised with leaders in the field presenting their work. Interaction with the delegates aid in building crucial networking opportunities and work relationships. The international arena is packed with new technology development allowing us the opportunity to learn and improve our own understanding of various concepts.

This conference proved to be an invaluable learning experience and I thank the NRF for this opportunity and for providing me with the funding to attend this conference.  The exposure to conferences, especially those in the international arena, aid in the development of students and contribute to the quality of research that is conducted at UCT.

References:

1. VLPNPV website

(http://www.meetingsmanagement.co.uk/index.php?option=com_content&view=article&id=33&Itemid=83)

2.  Personal notes taken at the conference

Scientists Find Compound That Helps HIV, Flu Vaccines – Health News – redOrbit

28 August, 2012

See on Scoop.itVirology News

“Oxford University scientists have discovered a compound that greatly boosts the effect of vaccines against viruses like flu, HIV and herpes in mice.”

 

Well, no, theyt haven’t: what they HAVE done is find that a very well known chemical has activity as an adjuvant – and very strong activity, it appears.

 

“The Oxford University team found that PEI, a standard polymer often used in genetic and cell biology, has strong adjuvant activity.
redOrbit (http://s.tt/1lPjE)”

 

It is also useful as a mucosal adjuvant, which is very useful for intranasal / oral vaccination strategies.

See on www.redorbit.com

PLoS Pathogens: ADCC Develops Over Time during Persistent Infection with Live-Attenuated SIV and Is Associated with Complete Protection against SIVmac251 Challenge

24 August, 2012

See on Scoop.itVirology and Bioinformatics from Virology.ca

“Here we show that live-attenuated SIV induces progressive increases in ADCC over time, and that the development of these antibodies is dependent upon the persistent replication of the vaccine strain. In two different experiments, the animals immunized with live-attenuated SIV that remained uninfected after pathogenic SIV challenge had higher measures of ADCC than those that became infected. Our results suggest that antibodies contribute to protection by live-attenuated SIV, and that persistent stimulation of antibody responses may be essential for HIV-1 vaccines to induce high ADCC activity.”

 

Shit HOT results, in that they demonstrate that – as some have said repeated ly over years – that neutralising Ab are NOT necessarily the Holy Grail, and that ADCC and other mechanisms are also really important.  Good Stuff…B-)

See on www.plospathogens.org

Vaccination with Adenovirus Serotypes 35, 26, and 48 Elicits Higher Levels of Innate Cytokine Responses than Adenovirus Serotype 5 in Rhesus Monkeys

24 August, 2012

See on Scoop.itVirology and Bioinformatics from Virology.ca

“These data demonstrate that Ad35, Ad26, and Ad48, which utilize CD46 as their primary cellular receptor, induce significantly greater innate cytokine responses than Ad5, which uses the coxsackievirus and adenovirus receptor (CAR). These differences in innate triggering result in markedly different immunologic milieus for the subsequent generation of adaptive immune responses by these vaccine vectors.”

 

Important news for the vectored vaccine community in general, and for HIV vaccine in particular: Ad5 was the vehicle of choice; now it looks as though it shouldn’t be.

 

Adenovirus graphic courtesy of Russell Kightley Media

See on jvi.asm.org

Gag-Specific Cellular Immunity Determines In Vitro Viral Inhibition and In Vivo Virologic Control following Simian Immunodeficiency Virus Challenges of Vaccinated Rhesus Monkeys

24 August, 2012

See on Scoop.itVirology News

“We observed that CD8+ lymphocytes from 23 vaccinated rhesus monkeys inhibited replication of SIV in vitro. Moreover, the magnitude of inhibition prior to challenge was inversely correlated with set point SIV plasma viral loads after challenge. In addition, CD8 cell-mediated viral inhibition in vaccinated rhesus monkeys correlated significantly with Gag-specific, but not Pol- or Env-specific, CD4+ and CD8+ T lymphocyte responses. These findings demonstrate that in vitro viral inhibition following vaccination largely reflects Gag-specific cellular immune responses and correlates with in vivo virologic control following infection. These data suggest the importance of including Gag in an HIV-1 vaccine in which virologic control is desired.”

 

In other words: having Gag or a gag gene included in a vaccine against SIV given to monkeys was more important than having Pol or Env when it came to control of virus replication – although, as has been shown elsewhere, Env responses are important for protecting against acquisition.  This has important implications for human vaccines – although “monkeys aren’t men, and mice lie” – and in particular for the South African SAAVI vaccines, which elicit quite good Gag-specific cellular responses.

 

We wait in hope.  Graphic showing immune cells associated with HIV control courtesy of Russell Kightley Media.

See on jvi.asm.org

What’s Causing the Spike in HIV Infection in Old Chinese Men? – Business Insider

23 August, 2012

See on Scoop.itVirology News

China DailyWhat’s Causing the Spike in HIV Infection in Old Chinese Men?

See on www.businessinsider.com

Papillomavirus and HIV: a nasty combination

17 August, 2012

I started working on human papillomaviruses (HPVs) some 22 years ago, back at the dawn of PCR: I helped my then-new major collaborator (and wife of 2 years), Anna-Lise Williamson, design some degenerate primers for amplifying as wide a range as possible of high-risk HPVs from cervical biopsy samples.  These worked pretty well, and are still highly useful for the purpose, despite the many novel types found since then.

We went on to do another two papers together on looking at variation and typing of HPVs via PCR and and sequencing, then took a deviation into making candidate vaccines for HPV and HIV.  Anna-Lise carried on with surveilling for HPVs, however, and has ended up with a WHO Regional Laboratory for HPV work.  She also started working on HPV infections in HIV-infected women: work on a study cohort showed that while HIV-free women usually had only 1 HPV type, the 109 HIV-infected often were infected with multiple HPV types.  In association with Anna Salimo in my lab, we started a deep sequencing pilot study on the sample with the most HPVs.  This turned this into a regional study, with help on assembling and interpreting sequence data from Prof Johan Burger’s lab at the University of Stellenbosch, and it was revelatory: while a commercial kit could detect 12 HPV types in one sample, next-gen sequencing found 16.

We went on to do PCR on all 109 samples in the cohort with specific primers for the types not found by the kit, and showed prevalences up to 15% in the HIV-infected group.  This is an important result, because otherwise-innocuous HPV types that do not show up in normal women, may well be associated with disease in the HIV-infected – and will probably not be protected against by the current HPV vaccines.

We continue to do work on these samples, and it will be very interesting to see what the new methodologies show up.  Especially as sequencing becomes cheaper, and we can do more samples…!  Meantime, we have published the pilot study:

Next-generation sequencing of cervical DNA detects human papillomavirus types not detected by commercial kits

Tracy L MeiringAnna T SalimoBeatrix CoetzeeHans J MareeJennifer MoodleyInga I HitzerothMichael-John FreeboroughEd P Rybicki and Anna-Lise Williamson

Virology Journal 2012, 9:164 doi:10.1186/1743-422X-9-164

Published: 16 August 2012

Abstract (provisional)

Background

Human papillomavirus (HPV) is the aetiological agent for cervical cancer and genital warts. Concurrent HPV and HIV infection in the South African population is high. HIV positive (+) women are often infected with multiple, rare and undetermined HPV types. Data on HPV incidence and genotype distribution are based on commercial HPV detection kits, but these kits may not detect all HPV types in HIV + women. The objectives of this study were to (i) identify the HPV types not detected by commercial genotyping kits present in a cervical specimen from an HIV positive South African woman using next generation sequencing, and (ii) determine if these types were prevalent in a cohort of HIV-infected South African women.

Methods

Total DNA was isolated from 109 cervical specimens from South African HIV + women. A specimen within this cohort representing a complex multiple HPV infection, with 12 HPV genotypes detected by the Roche Linear Array HPV genotyping (LA) kit, was selected for next generation sequencing analysis. All HPV types present in this cervical specimen were identified by Illumina sequencing of the extracted DNA following rolling circle amplification. The prevalence of the HPV types identified by sequencing, but not included in the Roche LA, was then determined in the 109 HIV positive South African women by type-specific PCR.

Results

Illumina sequencing identified a total of 16 HPV genotypes in the selected specimen, with four genotypes (HPV-30, 74, 86 and 90) not included in the commercial kit. The prevalence’s of HPV-30, 74, 86 and 90 in 109 HIV positive South African women were found to be 14.6 %, 12.8 %, 4.6 % and 8.3 % respectively.

Conclusions

Our results indicate that there are HPV types, with substantial prevalence, in HIV positive women not being detected in molecular epidemiology studies using commercial kits. The significance of these types in relation to cervical disease remains to be investigated.

I thank Russell Kightley Media for use of the HPV and cervical cancer graphic.


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