Human papillomavirus vaccines made in plants: a review Ed Rybicki Dept MCB / IIDMM University of Cape Town The Virus "HPV-16 genome organization" by Xmort en:wiki. Licensed under GFDL via Wikimedia
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File:HPV-16_genome_organization.png#/media/
File:HPV-16_genome_organization.png
Isometric unenveloped par2cles, ~60 nm diameter, T=7 symmetry ~8 kbp circular dsDNA genome Capsid has 72 pentamers of 55 kDa L1 major capsid protein; 12-­‐72 copies of 55 kDa L2 E6, E7 oncoproteins bind p53 and Rb, prevent apoptosis and promote cell cycle progression – expressed in tumours Cervical Cancer and HPV §  Cervical cancer is the second most prevalent cancer in women worldwide and the most common cancer in developing countries (IARC 1999) §  in 2001 women in developing countries accounted for ~85% of both annual cases of cervical cancer (~500 000 cases worldwide) and annual deaths from cervical cancer (~300 000 worldwide) §  Sub-­‐Saharan Africa and SE Asia are the areas of highest cervical cancer mortality rates §  100% of cervical cancers are caused by high-­‐risk HPVs (at least 50% HPV-­‐16) ProphylacPc Vaccines •  HPV prophylac2c vaccines are based on VLPs made by recombinant expression and assembly of the major capsid protein L1 •  Merck’s yeast-­‐made Gardasil targets HPV-­‐16 and HPV-­‐18 found in 70% of cervical cancer cases and genital wart-­‐causing viruses HPVs −6 and −11 •  GSK’s insect cell-­‐made Cervarix contains only HPVs −16 and −18. •  They are already blockbuster vaccines, with annual sales close to US$1 billion as of 2012 •  Merck’s nonavalent VLP vaccine was approved by FDA in December 2014 – may prevent >80% of Ca cervix •  “ProtecPon against the targeted HPV types has been found to last for at least 8 years with Gardasil and at least 9 years with Cervarix“ -­‐ NIH’s NCI Problems with HPV vaccines: •  Cost: 2-­‐3 doses needed, lowest price $4.50/dose for 2-­‐4-­‐
valent vaccines ONLY with GAVI in SOME developing countries •  Virus coverage : developing country prevalence differences and different spectrum of HPVs in HIV+ people means inadequate coverage •  Non-­‐therapeuPc: neither Gardasil nor Cervarix are effec2ve against pre-­‐established infec2ons or cancers •  Next-­‐generaPon mul2valent or chimaeric or L2-­‐based vaccines will not address this •  NEED CHEAP, PROPHYLACTIC AND THERAPEUTIC VACCINES Plant-­‐made HPV vaccine candidates: prophylacPc Year
Landmark
2001*
Oral immunogenicity of HPV L1 coadministered with E coli enterotoxin mutant
R192G or CpG DNA (Gerber et al., 2001)
2002
US6444805 claims HPV-16 and -18 L1 and L1+L2 VLPs made in transgenic tobacco
and use as a vaccine (Sohn et al.) 2003*
Oral immunogenicity of HPV-11 L1 VLPs expressed in transgenic potato;
immunogenicity of oral and injected HPV-16 L1 VLPs from transgenic tobacco
(Warzecha et al.; Biemelt et al., Varsani et al., 2003)
2006*
Transient expression of HPV-16 L1 protein in Nicotiana benthamiana using an
infectious tobamovirus vector; immunogenicity in rabbits (Varsani et al., 2006)
2006*
Proof of efficacy of plant-produced papillomavirus vaccines in a rabbit model:
CRPV L2 epitopes on TMV; CRPV L1 via transgenic tobacco and rTMV infection
(Palmer et al.; Kohl et al., 2006)
2007* 2008
High-level transient expression of HPV-16 L1 protein and VLPs in N benthamiana
(Maclean et al., 2007) and via transplastomic expression in tobacco (
Fernandez-San Millan et al., 2008), and proof of neutralising antibody production in
mice for both
Results of CRPV Challenge: 1st proof of efficacy of a plant-­‐
produced papillomavirus L1 protein vaccine 18
16
Papilloma size mm
14
12
Controls: BCG-­‐rota (-­‐ve) and CRPV VLPs (+ve) 10
8
6
4
TMV 2
0
14
21
28
35
42
49
Tr 56
63
Days after CRPV challenge
TMV-derived CRPV L1
Transgenic plant-derived CRPV L1
Control group
CRPV VLPs
70
HPV vaccine candidates: prophylacPc & therapeuPc 2011
Transplastomic expression of capsomere-forming HPV-16 L1 fused with E coli LTB
as a built-in adjuvant (Waheed et al., 2011)
2012
Successful expression of HPV-8 and Bovine papillomavirus L1 VLPs in plants via
agroinfiltration and demonstration of immunogenicity (Matic et al., Love et al., 2012)
2012
Transgenic expression of HPV-16 L1 with E coli LTB and oral immunisation elicits
increased intestinal mucosal IgA responses to L1 (Hongli et al., 2012)
2013*
High-yield plant transient expression of chimaeric L1::L2 VLPs and proof of
increased breadth of neutralising immune response (Pineo et al., 2013)
2014
Plant expressed HPV-16 L1 with C-terminal string of E6 and E7 T-cell epitopes is
viable prophylactic/therapeutic vaccine candidate (Monroy-Garcia et al., 2014)
2002
Plant-derived HPV 16 E7 oncoprotein induces immune response and specific
tumour protection in mice (Franconi et al., 2002)
2007 2012
Plant production, scale-up and protective efficacy in mouse model of therapeutic
E7GGG-LicKM fusion protein vaccine (Massa 2007; Venuti 2009; Buyel 2012)
2013
Production and proof of efficacy in mouse tumour model of soluble E7GGG
therapeutic vaccine in transplastomic Chlamydomonas reinhardtii (
Demurtas et al., 2013)
2014*
Proof of yield increase and efficacy in a mouse tumour model of shuffled E7 protein
fused to Zera® peptide (Whitehead et al., 2014)
Results of Tumour Challenge in Mice: Mice were inoculated with 0.5  ×  106 C3 tumour cells to induce tumours
and subsequently injected with vaccine (5 µg protein or 100 µg DNA in
100 µl) in two sites per animal when the tumours were clearly
palpable.
Whitehead et al., 2014, http://www.biomedcentral.com/1471-2407/14/367
Yield increase for HPV-­‐16 L1, 2000-­‐2006 ug/kg
1000000 100000 10000 1000 100 10 1 2 000 UNSTABLE
250,000 no localisa2on plas2d Expression and localisa2on of codon op2mised HPV-­‐16 L1 H16.V5 capture H16.J4 capture GFP capture 650 600 4.5% TSP 500 1.6 1.4 14.9% TSP 450 1.2 1 400 350 0.8 300 250 0.6 200 0.4 150 100 0.2 50 0 0 pTRA-­‐ SAL1 pTRAERH-­‐ pTRACTP-­‐ SAL1 SAL1 Wild-­‐type pTRA-­‐ SYNL1 pTRAERH-­‐ pTRACTP-­‐ pTRA-­‐HL1 pTRAERH-­‐ pTRACTP-­‐ pTRACTP-­‐ SYNL1 SYNL1 PlanPsed HL1 HL1 HL1ΔC22 Humanised Plant control GFP (OD492nm) mg of L1 per kg plant material 550 17.1% TSP Importance of antigen sequence
•  L1 chimaeras = L2 and E7
epitopes incorporated into
HPV-16 L1 & displayed on the
surface, made in insect cells
•  Pineo et al. (2013): epitope sequences
derived from HPV-16 L2 amino acid
108–120, 56–81 or 17–36, in plants
•  Got cross-neutralisation of
different types
10000
4.96%
1000
100
0.22%
0.34%
0.20%
0.29%
0.07%
10
0.02%
tr
ol
co
n
Ss
N
/L
2
/E
7
H
M
L1
/E
7
/L
2
L1
L1
/E
7
H
M
/E
7
L1
88
)
PV
(1
-
L1
/L
2
B
/L
2
(1
7
-3
6
)
-8
1
L1
L1
/L
2
(5
6
812
(1
0
)
1
/L
2
L1
19.9%
14.7%
0)
Log protein yield
(mg protein / kg plant tissue)
Sequence: Small differences matter!
Maximum protein yields obtained for the chimaeras co-expressed with the NSs silencing
suppressor and targeted to the chloroplast. Protein yields expressed as a % TSP are
indicated above the bars.
Fusion partner is important
1.1 g/kg vs undetectable
Whitehead et al., 2014, http://www.biomedcentral.com/1471-2407/14/367
Lessons learned in 15 years •  HPV/PV VLPs can be made at high yield in plants •  Codon opPmisaPon is important •  Intracellular compartment may be important •  Plant-­‐made PV VLPs highly immunogenic, elicit neutralising an2body responses, and are effecPve •  Injectable and orally-­‐delivered VLPs are immunogenic •  Second-­‐genera2on L2-­‐containing vaccines are feasible •  L2 pep2des as chimaeras or L2 protein in par2cles •  Plant-­‐made therapeu2c vaccine candidates based on E7 protein are viable and effecPve •  PepPde presentaPon or mulPmerisaPon most effec2ve •  N benthamiana extracts are self-­‐adjuvanPng – an advantage?? What about the future? •  The BRU is working with Medicago to make mulPvalent plant-­‐
made VLP-­‐based vaccine •  Prospect of VLP-­‐based vaccines for epidermodysplasia verruciformis and bovine papillomas? •  Use of PV VLPs as scaffold for presentaPon of other epitopes eg: L2 epitopes, flu M2e epitope •  Clinical trial of plant-­‐made E7-­‐based vaccine candidates in cancer paPents? Eg: Zera-­‐E7SH; Saporin E7GGG? •  CombinaPon prophylacPc / therapeuPc vaccines could be trialled – eg: L1 with E6 / E7 epitopes; HPV pseudovirions + DNA vaccine (Renate Lamprecht) With special thanks to the HPV green team, present and past: A.-­‐L. Williamson, Inga Hitzeroth, Ann Meyers, Alta van Zyl, Renate Lamprecht, Megan Hendrikse, Mark Whitehead, Guy Regnard, Aleyo Chabeda Sue Huddy, Paul Kennedy, Benjamin Singo, James Maclean, Arvind Varsani, Thomas Kohl, Ramon Pereira Cathy Pineo And to: Rainer Fischer, Thomas Rademacher -­‐ RWTH Aachen University Neil Christensen – Univ Pennsylvania MarPn Muller – DKFZ, Heidelberg Peter Oelschlager – Univ Konstanz PRF