“Evaluation of immune response to Measles vectored & Pichica Pastoris expressed recombinant HPV vaccine formulations ” Gupta Gaurav, Gianninò Viviana, *Rishi Narayan, Glueck Reinhard. Vaccine technology center, Cadila Healthcare Ltd., Ahmedabad, India;*Amity Institute of Virology and Immunology, Noida,India Introduction – Human Papilloma Virus (HPV) Papilloma viruses (HPVs), comprising an heterogeneous group of more than 80 epitheliotropic genotypes, are a large family of small double-stranded DNA viruses that infect squamous epithelia. Most of the HPV types produce benign lesions, a small subset of genotypes is strongly associated with the development of high-grade squamous intraepithelial lesions and cervical cancer. HPV is responsible for approximately half a million new cervical cancer cases every year, as HPV DNA of high-risk genotypes can be found in more than 95% of these cancers. HPV type 16 and 18 are associated with about 70% cervical cancer cases. Introduction – Measles virus as recombinant vaccine vector Measles virus (MeV), an enveloped virus with a negative-strand RNA genome belonging to the family Paramyxoviridae is the causative agent of a highly contagious disease. Live attenuated MeVs are safe, yet highly efficient childhood vaccines, inducing long-lived immunity after a single immunisation dose. Methods exist to rescue attenuated MeV variants from cloned cDNA, also allowing the insertion of heterologous genes in different MeV genome positions, resulting in stable expression at different levels of the transgenic proteins. Recombinant MeVs have been shown in animals to induce comparable immunity against MV proteins and transgenic antigens. We use the reverse genetics system based on recombinant MeV Edmoston-Zagreb (EZ) vaccine strain to create MeV carrying transgenes encoding main antigens from Human Papilloma Virus (HPV) to develop new recombinant vaccine, useful in the prophylaxis of cervical carcinoma. V C T7 N H F L M P T7t d 3 2 1 100 % 1.5 % mRNA transcription gradient Rescue of recombinant viruses employs the human cell line 293T, co-transfected with helper plasmids, providing the P, N and L viral function, recombinant MeV antigenomic plasmid and a plasmid enconding for T7 polymerase. Recombinant viruses rescued are analyzed for genetic stability and transgene expression in cell cultures, and for immunogenicity in CD46 transgenic mice. 293 cell line with T7 RNA pol N and P Cotransfection Syncytia formation Virus rescue p(+)MV T7 pEMCLa EMC IRES L -Production of HPV virions is strictly linked to well-differentiated epithelial cells. -L1 and L2 genes, encoding respectively the major capsid protein and the minor capsid protein, are expressed under cis-acting negative-regulatory control, which is type-dependent and cell-differentiation-dependent (Sokolowski et al., J.Virol 1998,72:1504). E6 encodes a 16-19 kD cell-trasforming protein, acting also as DNA-binding protein. It forms a complex with p53 promoting its degradation, thus interfering with the cell-cycle. E7 encodes a 10-14 kD cell-trasforming protein, acting with E6 to immortalize squamous epithelial cells. It binds pRB, p107 and p130. Steps of the work Pichia Pastoris based HPV vaccine Gene synthesis for yeast codon optimized HPV 16 L1 and HPV 18 L1 and cloning in E coli followed by transfection in Pichia Pastoris Selection of clones based on gene copy no by Q PCR and expression analysis by SDS & western blot Characterized for VLP formation by Electron microscopy Fermentation, purification by column and/or ultracentrifugation, formulation with Alum Immunogenicity testing in female Balb c/mice with commercial vaccine (Cervarix) and ELISA for seroconversion Expression of the HPV major capsid gene L1 is sufficient for generation of virus-like immunogenic particles (VLPs), while the HPV minor capsid protein L2 confers cross-protection against different HPV types. L1 -Type-specific antigen. -No cross neutralisation. -Induces high neutralising-antibody titres. L2 -Cross-protection against unrelated types. -Sub-dominant when associated with L1 in VLPs. -Epitopes localisation: 17-36, 65-81, 94-122, 131-151, 151-170 (in rabbit) -Induces low neutralising-antibodies titres in comparison to L1. HPV16L1 Immunogenicity results To develop a MeV-based HPV vaccine, we generate recombinant viruses expressing L1 protein of HPV16 and HPV18, L2 protein of HPV16 and 18, in full-length or truncated forms. Transgenes, obtained by PCR or synthetic, are cloned by restriction with BssHII and AatII in two positions of the MeV antigenome, under the control of a T7 promoter. Introduction- Pichia Pastoris based HPV vaccine Currently, two HPV VLP vaccines are available on the market. Both vaccines, Gardasil® (Merck) and Cervarix®(GSK), provide prophylaxis against HPV types 16 and 18, which are prevalent in 70% of cervical cancer cases. Gardasil® further provides protection against HPV types 6 and 11 in an attempt to reduce the incidence of genital warts by 90%. Unfortunately the costs of these vaccines are prohibitively expensive for general distribution in developing countries. The purpose of the present work is therefore to investigate alternative HPV vaccines with serotypes of relevance to the developing world, making use of alternative microbial production systems such as Pichia pastoris for the production of VLP vaccine candidates. Pichia has the many advantages as expression host: first, extremely high yields of intracellular proteins; second, very high levels of secretion into an almost protein-free medium; third, ease of fermentation to high cell density; and fourth, genetic stability and scale-up without loss of yield. It is proving valuable in producing large amounts of protein and reducing cost of vaccine production. Therefore we selected HPV 16 L1 and HPV 18 L 1 gene for cloning and expression as VLP’s for vaccine production. Sr no Sample Dilution Total No. of animals No of animals with Seroconversion 1 Reference HPV16L1 (Cervarix) 1:100 10 7 1:500 8 1:5000 4 2 HPV16L1 9 3 Reference HPV18L1 (Cervarix) 6 HPV18L1 5 M Ref 16 L1 batch 1 batch 2 batch 3 M Ref 16 L1 batch 1 batch 2 batch 3 HPV18L1 Steps of the work Measles vectored Vaccine Use the attenuated MeV Edmoston vaccine strain to create recombinant MeV viruses, using HPV16L1, HPV18L1, HPV16L2 and HPV18L2 as transgenes, respecting the “rule of six”; Use of 293T cells for the rescue of recombinant MeV antigenomic plasmids in a five-plasmids trasfection system; Analysis of the recombinant viruses by Western blot,, IFA , serial passages in cells, and growth kinetics; Immunogenicity testing in CD46 transgenic mice. batch 1 batch 2 batch 3 ref 18 L1 M batch 1 batch 2 batch 3 ref 18 L1 M AatII T7 d/TF BssHII Transgene P2 HPV16 L1 full-length HPV18 L1 HPV16 L2 full-length HPV18 L2 full-length HPV16 L2 (N-terminal) HPV18 L2 CMV-IRES Peptide polilinker Transgene(s) L1-based L2-based HPV18 L1 full-length N P M F H L Immunofluorescence assay for HPV-L1 protein. Vero cells monolayers infected with MV “empy” (A-C) and rMV-HPV16L1 (B-D). Cells were stained with a monoclonal anti-HPV16-L1 antibody subsequently with a goat anti-mouse coupled to FITC (green) (C-D) A B C D Western blot for expression of HPV16-L1; 1. lysed cells infected with MV “empy”; 2. lysed cells infected with rMV-HPV16-L1. 124 34 80 25 41 55 L1 1 2 1.E+00 1.E+01 1.E+02 1.E+03 1.E+04 1.E+05 HPV ELISA assay HPV neutralisation assay Titer MV2EZ-HPV16-L1 animal #1 MV2EZ-HPV16-L1 animal #2 MV2EZ-HPV16-L1 animal #3 MV2EZ-HPV16-L1 animal #4 MV2EZ-HPV16-L1 animal #5 MV2EZ-HPV-16L1 animal #1-#5 Pool Pooled sera of MV naive animals Pooled sera of MVEZ animals ELISA and neutralization assay (performed from Dr. Schiller at the NIH) of sera from CD46 mice immunised with rMV-HPV16-L1.