María G. Guzmán Instituto Medicina Tropical Habana, Junio Dengue Vaccine Development Cuban studies

Transcription

1 María G. Guzmán Instituto Medicina Tropical Habana, Junio 2004 Dengue Vaccine Development Cuban studies

2 Four viruses Dengue Virus Ab ADE Hyphotesis Fc RECEPTOR CELL {Four Four viruses {ADE {No Animal Model {Long Protection to the four viruses {Disease of the development world {Non enough financial support for research {Pathogenesis mechanisms? Interval of Infection: 4 years versus 20 years Todos S. Halstead

3 Dengue Vaccine Challenges IPK Long lasting protection to the 4 serotypes No ADE Useful for children and adults Non expensive Introduction of the

4 Strategies Dengue Vaccine Conventional (inactivated & attenuated) Non Non recombinant (structural & non structural purified proteins, synthetic peptides) Recombinant subunit (E. Coli, Baculovirus, Yeast) Recombinant vaccinia Dengue vaccines Infectious cdna clone (chimeric( vaccines) DNADNA IPK

5 Current Vaccine Candidates Live attenuated vaccines Mahidol Univ Aventis Pasteur Phase 3 in plan Walter Reed Army Institute GSK Phase 1 & 2 Chimeric Vaccines Acambis (YF/Den) Aventis Pasteur Phase 1 & 2 CDC (Den2/Den) Preclinial studies NIH (Den4/Den) Phase 1

6 Current Vaccine Candidates DNA vaccines WRAIR Den 1 Preclinical studies (monkey studies) Recombinant Vaccines Hawaii Biotech Den1-4 Preclinical S. monkey studies IPK/CIGB Den4/yeast Preclinical S monkey studies Dom B

7 Cuban IPK/CIGB Studies Objectives: To evaluate the usefulness of recombinant E protein as a vaccine candidate Previously, virus attenuation studies and research on prm/m proteins have been developed

8 Serial Passage of Den 2 & Den 4 viruses in Primary Dog kidney Den 2, passage level 53 & Den 4, passage level 48/49 Small plaque size Temp sens C Reduced neurovirulence in mice No CPE in LLCMK & C636 cells Infective titers Studies in course to evaluate virulence markers Alvarez et al., Int J Inf Dis 01, Pupo et al., non published

9 T and B cell epitope definition on prm/m protein prm glycoprotein, 18kDa precursor of M protein 7kDa Co-transport of prm/e heterodimer through exocytic pathway E protein requires co-synthesis of prm Mature virions contain M prot

10 To determine protective epitopes on prm/m protein that could be included in a vaccine Prediction of antigenic regions & T cell epitopes of prm protein if dengue virus amino acid flexibility amino acid flexibility Multiple sequence alignement corresponding to protein pre-(non M) of flaviviruses dengue pre- Variability dengue M Variability Antigenic peptides proposed in pre- M protein of dengue virus ; den1a -FHLTTRG---GEPTLIVSKQERGKSLLFKT-SAGVNMCTLIAMDLGELCEDTMTYK 2; den1c -FHLTTRG---GEPTLIVSKQERGKSLLFKT-SAGVNMCTLIAMDLGELCEDTMTYK 3; den18 -FHLTTRG---GEPTLIVSKQERGKSLLFKT-PAGVNMCTLIAMDLGELCEDTMTYK 4; den1w -FHLTTRG---GEPHMIVSKQERGKSLLFKT-SAGVNMCTLIAMDLGELCEDTMTYK 5; den1s -FHLTTRG---GEPHMIVSKQEREKSLLFKT-SVGVNMCTLIAMDLGELCEDTMTYK 6; den2u -FHLTTRN---GEPHMIVSRQEKGKSLLFKT-EDGVNMCTLMAMDLGELCEDTITYK 7: den2cu* -FHLTTRN---GEPHMIVMRQEKGKSLLFKT-GDGVNMCTLMAMDLGELCEDTITYK 8; den3 -FHLTSRD---GEPRMIVGKNERGKSLLFKT-ASGINMCTLIAMDLGEMCDDTVTYK 9; den27 -FHLTTRN---GEPHMIVSRQEKGKSLLFKT-EVGVNMCTLMAMDLGELCEDTITYK 10; den26 -FHLTTRN---GEPHMIVSRQEKGKSLLFKT-EDGVNMCTLMAMDLGELCEDTITYK 11; den2p -FHLTTRN---GEPHMIVSRQEKGKSLLFKT-KDGTNMCTLMAMDLGELCEDTITYK 12; den2j -FHLTTRN---GEPHMIVGRQEKGKSLLFKT-EDGVNMCTLMAIDLGELCEDTITYK 13; den2d -FHLTTRN---GEPHMIVSRQEKGKSLLFKT-EDGVNMCTLMAIDFGELCEDTITYK 14; den4 -FSLSTRD---GEPLMIVAKHERGRPLLFKT-TEGINKCTLIAMDLGEMCEDTVTYK 15; wnv -VTLSNFQ---GKVMMTVNATDVTDVITIPT-AAGKNLCIVRAMDVGYLCEDTITYE 16; jaev1 -MKLSNFQ---GKLLMTINNTDIADVIVIPT-SKGENRCWVRAIDVGYMCEDTITYE 17; jaevn -MKLSNFQ---GKLLMTVNNTDIADVIVIPNPSKGENRCWVRAIDVGYMCEDTITYE 18; jaev5 -MKLSNFQ---GKLLMTINNTDIADVIVIPT-SKGENRCWVRAIDVGYMCEDTITYE 19; jaevj -MKLSNFQ---GKLLMTINNTDIADVIVIPT-SKGENRCWVRAIDVGYMCEDTITYE 20; kunjm -VTLSNFQ---GKVMMTVNATDVTDIITIPP-AAGKNLCIVRAMDVGHMCDDTITYE 21; yefv2 -VTLVRKN---RWLLLNVTSEDLGKTFSVGT-----GNCTTNILEAKYWCPDSMEYN 22; yefv1 -VTLVRKN---RWLLLNVTSEDLGKTFSVGT-----GNCTTNILEAKYWCPDSMEYN 23; yefv8 -VTLVRKS---RWLLLNVTSEDLGKTFSVGT-----GNCTTNILEAKNWCPDSMEYN 24; stevm -LQLSTYQ---GKVLMSINKTDAQSAINIPS-ANGANTCIVRALDVGIMCKDDITYL 25; mvev ALKLSTFQ---GKIMMTVNATDIADVIAIPT-PKGPNQCWIRAIDIGFMCDDTITYE 26; tbevw -MTLAATVRKERDGSTVIRAEGKDAATQVRVE---NGTCVILATDMGSWCDDSLSYE 27; tbevs -VTLAATVRKERDGTTVIRAEGKDAATQVRVE---NGTCVILATDMGSWCDDSLTYE 28; liv -MTLAATVRKEGDGTTVIRAEGRDAATQVRVE---NGTCVILATDMGSWCDDSLSYE 29; lanvt -MCLTATVRRERDGSMVIRAEGRDAATQVRVE---NGTCVILATDMGSWCDDSLAYE 30; lanvy -MCLTATVRRERDGSMVIRAEGRDAATQVRVE---NGTCVILATDMGSWCDDSLAYE PHD sec EEEE EEEEEEEE EEEEE EEEEEEEE EEEE PHD acc eebeeee---bee bbbeeeeebeebbbebeeebebbbbbbbbbbbebbeebbebe ID * * * * * Vazquez et al., Vaccine 02 C O D E P R O T E IN S E Q U E N C E R E G IO N B 19-6 pr LTTRNGEPHMIVMRQEKGKSLLFKTGDGV 3-31 B 20-2 pr CEDTITYKCPLLRQNEPEDIDCW B 19-5 pr RQNEPEDIDCW CNSTSTW VTYGTCTTTGEHRREKRS B 20-1 pr NSTSTW VTYGTCTTTGEHRREKRSV B 20-3 M LETRTETW MSSEGAW KHAQRIE

11 utralizing Ab titer of anti-peptide mice sera Proliferative response of antigen- specific T cells Anti-peptide Neutralizing titer for each serotype D 1 D 2 D 3 D 4 B /100 1/180 1/60 1/160 B 20-2 NR <1/10 NR NR B 19-5 NR <1/10 NR NR B 20-1 NR <1/10 NR NR B /110 1/80 1/80 1/80 BSA <1/10 <1/10 <1/10 <1/10 Pre-immune sera <1/10 <1/10 <1/10 <1/10 peptides B20-3 B20-2 B19-5 B20-1 B stimulate index CC Dengue 2 40ug/ml Shyntetic peptides Mice immunization Survival (%) Mice protection after challenge days post-infection Protection/D2 Nt/virus LPA/D2 Vazquez et al., Vaccine 2002 B19-6 B20-1 B19-5 B20-2 B20-3 BSA

12 prm/m Dengue 2 Mice sera antipeptide Pept. ELISA NT* Protection LPA** * Nt Ab titer 1/80-1/160 to the 4 viruses **To D2 One epitope able to induce neutralization & protection in mice Vazquez et al., Vaccine 2002

13 Recombinant strategy E protein Evaluation of Dengue 4 virus envelope glycoprotein expressed in Pichia pastoris Evaluation of B domain of Dengue envelope protein expressed in E coli

14 E protein contains the major epitopes responsible for eliciting neutralizing antibodies Limited reported experiences using yeast as expression system cdna encoding the Den 4 E protein was expressed in Pichia pastoris. P F e The E gene was truncated at the carboxy terminus to remove the hydrophobic membrane anchor segment in order to maximize the secretion of the protein

15 Sera Mice immunization w/e4rec Mice sera E4rec has a MW of 60 kda, glycosylated ELISA HIH NT(D4) / / / / /20 - It induces both humoral & cellular immune response in immunized mice and protection after challenge E4rec 13/15 (86.6%) p< Control 0/15 (0%) Muné et al., Archives Virol, 2003 Mice Protection

16 Evaluation of E4rec in Macaca fascicularis monkeys Group 1: four immunizations 100ug of E4rec/alum Group 2: control IPK/CIGB Challenge: 10 5 pfu Den 4 virus Serological & virological studies Guzmán et al., AJTMH, 2003

17 Neutralizing antibody titres in immunized monkeys at days 0 and 30 after challenge with dengue 4 virus. Erec GMT D D D D ControlD D Results expressed as the reciprocal dilutions of the PRNT50 All animals developed Nt Abs (>1/20) to Den 2 virus after 30 days of challenge. One monkey showed 2.8-fold rise in Nt Ab titer. The others 7.3 and 21.3-fold increase Day 30 p<0.05 IPK/CIGB

18 % Viremia Percentage of viremia in immunized monkeys and controls One vaccine immunized monkey was protected from viremia while the other two were partially protected with viremia lasting 3 and 7 days IPK/CIGB Days postchallenge E4rec Control Control group developed viremia lasting 5, 7 and 13 days The mean duration of viremia in naive animals was 8.33 days while viremia in immunized animals lasted 3.33 days None of the animals developed sign of illness

19 E4rec was immunogenic in fascicularis monkeys One animal was fully protected & two partial protected (reducing viremia) No clinical signs or abnormalities in hematological tests were observed during immunization and challenge. Vaccinated animals showed anamnestic antibody responses upon virus challenge indicating successful priming by the recombinant vaccine. The fully protected monkey developed the lowest antibody titers after challenge showing the lowest fold rise in neutralizing antibodies.

20 Apparently, E4rec was not able fully to protect the vaccinated animals. Conformational differences between recombinant and native protein??? antigen presentation or the applied immunization schedule??? Neutralizing antibody is important for protection against dengue virus infection. However, the presence of neutralizing antibody in our animals did not correlate with protection from viremia. All vaccinated monkeys had the same neutralizing antibody titer (1/30) at the time of challenge however they were not protected in a uniform manner. Perhaps humoral or cellular immune response were not sufficient despite the rapid postchallenge increasing of neutralizing antibody.

21 Recombinant strategy E protein Evaluation of Dengue 4 virus envelope glycoprotein expressed in Pichia pastoris Evaluation of B domain of Dengue envelope protein expressed in E coli

22 Flaivirus envelope glycoprotein Dom B

23 Den 1 & Den 2 Dom B of E protein inserted within the structure of p64k meningococcal protein carrier Genetic constructions DomB inserted P64K NH 2 Dom B P64k P64k COOH Fused to C- terminus P64K NH 2 P64k Dom COOHB COOH Hermida et al.,biotechnol Appl Biochem 2004 Hermida et al., J Virol Methods, 2004 IPK/CIGB

24 Evaluation in mice of different genetic constructions Neutralizing Antibody Development Immunogen GMT Den2 PD3 (fus) 519 Den 1 PD10 (ins) 103 Den 1 PD11 (fus) 170 Hermida et al.,biotechnol Appl Biochem 2004 Hermida et al., J Virol Methods, 2004

25 % de ratones % de sobrevida sanos % de sobrevida Días posteriores al reto A Control (-) PD11 PD10 DEN Días posteriores al reto Control (-) PD11 PD10 DEN 1 Control (-) PD3 ins PD5 fus DEN 2 Den 2 Den 1 Survival of immunized mice Días posteriores al reto Hermida et al.,biotechnol Appl Biochem 2004 Hermida et al., J Virol Methods, 2004

26 Evaluation of Den 2 B domain (expressed in E coli) in Macaca fascicularis monkeys Group 1: PD5 (DomB-Cterm P64K) Group 2: PD3 (DomB-Nterm P64K) Group 3: P64k 50 ug/fa 3 animals/group 4 immunizations Rodriguez R et al, J Virol submitted

27 Protection of monkeys against challenge with Den 4 virus Immunogen Mean viremia % protected V. isolation PCR PD5 0 3 days (1) 100 PD3 1 day (0.33) 9 days (3) 66 Control 8 days (3) 17 days(6) 0 Tested by Virus isolation and PCR Rodriguez R et al, J Virol submitted

28 GMT PRNT prechall enge & 18 and 69 d ay s after chall enge PD5 PD3 CONTROL Nt Ab response after immunization Anamnestic responbse after challenge pre-reto 18 days 69 days R. Rodríguez et al., J Virology submitted

29 Main Results Development of specific Nt Abs after 3 and 4 doses Total Protection (pd5) after challenge Partial protection (pd3) after challenge Anamnestic response Fusion protein C > insert protein Protection after challenge Nt Abs development R. Rodríguez et al., J Virology submitted

30 Conclusion Studies in course in order to evaluate the usefulness of B domain as a vaccine candidate IPK

31 María G. Guzmán Rayner Rodríguez Mayra Muné Susana Vázquez Mayling Alvarez Rosmari Rodríguez Yudira Soto Rosa Ramírez Beatriz Sierra Ana B Pérez Delfina Rosario Maritza Pupo Gissel García Lídice Bernardo Irina Prado Sheyla Cabezas Teresita Serrano Gerardo Guillén Lissete Hermida Laura Lazo Rolando Paez Carlos López Ida Valdés Aida Zulueta Jorge Martín Raúl Espinosa Rafael Martínez Karelia Cosme IPK/CIGB

32 9no Curso Internacional de Dengue, Agosto, 2005