1 Pros and cons of DNA vaccines against chronic viral infections and the advantages of combining different vaccination approaches Maria Isaguliants Riga Stradins University, Riga, Latvia and Karolinska Institutet, Stockholm, Sweden
2 Eukaryotic cell barriers to the viral life cycle and types of subversion mechanisms that are used by the virus Bruggeman, L. A. Clin J Am Soc Nephrol 2007;2:S13-S19 Copyright ©2007 American Society of Nephrology
3 Bruggeman, L. A. Clin J Am Soc Nephrol 2007;2:S13-S19
4 Examples of the viruses causing chronic infections:HSV-1/HSV-2 CMV HCV HIV-1 HPV family Examples of the approaches to vaccine design
5 Vaccine Type Disease Live, attenuated vaccineMeasles, mumps, rubella, polio (Sabin vaccine), yellow fever Inactivated or “killed” vaccine Cholera, flu, hepatitis A, Japanese encephalitis, plague, polio (Salk vaccine), rabies Toxoid vaccine Diphtheria, tetanus Subunit vaccines Hepatitis B, pertussis, pneumonia caused by Streptococcus pneumoniae Conjugate vaccines Haemophilus Influenza type B, pneumonia caused by Streptococcus pneumoniae DNA vaccines In clinical testing Recombinant vector vaccines
6 Needles versus No needlesOral vaccines: polio, typhoid, cholera, rotovirus, nasal influenza Cutaneous vaccination: liquid-jet injection deliver antigen id, sc or im (Advantage-Langerhans cells in skin-requires lower dose of antigen. Disadvantage-blood transfer) Particle bombardment of the skin - epidermal powder administration Topical application to skin using adjuvants and or permeabilizing agents Problems with Needle Administration of Vaccines 750 million injection given annually Limitations -costs -needle phobia -accidental sticks -improper and unsafe use
7 Genetic vaccines – new technique, new hopeGenetic vaccines are based on DNA or RNA Encodes the vaccine antigen Stimulates both B and T cell responses Induces an immune response resembling that during a viral infection Över till de vacciner som jag jobbar med, en ny generation vacciner - de genetiska. G. Karlsson-Hedestam
8 The cells of the body act as vaccine factoriesDNA Presentation of protein fragments on MHC class I molecules Secreted protein protein translation mRNA transcription Poängen med genetiska vacciner är att kroppens celler själva producerar vaccinprotein. Cellerna tar upp plasmiden och börja producera proteinet som kodas I plasmiden. På detta sätt simulerar man en virusinfektion och man kan stimulera delar av immunförsvaret (det cellulära), som man inte gör när man vaccinerar med protein direkt. Muscle/skin/lymphoid cell G. Karlsson-Hedestam, SMI 2006
9 DNA vaccines PROS CONS
10 Efficacy: stepwise changes make a differenceFelber BK, Velantin A et al, 2014
11 Reduced number of consensus transcription factor-binding siteshttps://worldwide.promega.com/resources/product-guides-and-selectors/protocols-and-applications-guide/bioluminescent-reporters/
12 Manipulations with encoded antigens allowing retargeting of processing and presentationMORE PROS
13 Targeting of drug resistant HIV-1 reverse transcriptase (RT) to the lysosomal degradation pathwayIsaguliants M & Starodubova E, 2009
14 Potentiating immune response against drug resistant HIV-1 RT after its targeting to the lysosomal degradation pathway Specific IL-2 production Specific IgG response
15 Efficacy: stepwise changes make a differenceFelber BK, Velantin A et al, 2014
16 Ways of gene delivery 29G needles OR Microneedles Biojector(delivery by gas pressure) Electroporation Dermavax, Cellectis CUY21EDIT, BEX 16
17 Very efficient in (small) animal models, cheap, easy to produce, handle and store;Safe in small animals, larger species, humans Low immunogenicy in larger species; Difficult to enhance the efficacy in large species; Multiple clinical trials with little success. OLD CONS NEW CONS
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20 Recombinant virus as vaccinesVirus genome With recombinant DNA technique the microbial gene(s) are cloned into the attenuated virus Aattenuated virus (Pox-, Adeno-, Alfavirus etc.) Gene insert The recombinant virus infects cells and foreign genes are delivered and expressed as proteins Då plasmiderna inte ger upphov till så starkt immunsvar I människa som man skulle vilja utvecklar man strategier för att öka potensen. En trolig orsak till det låga svaret I människa är att leveransen av plasmider är oeffektiv och endast en bråkdel av plasmiderna tas upp. För att öka upptaget av vaccingenen använder man sig av rekombinanta virusvektorer. Vad man gör är att man använder sig av försvagade (attenuerade) virus som amn inte blir sjuk av och med hjälp av rekombinant DNA teknik klonar man in Vaccingener, I detta fall HIVgen I det försvagade virusets arvsmassa. När virus sen infekterar celler så får man en väldigt effektiv levereans av sin vaccingen.
21 VIRAL VECTORS CONS PROSViruses are naturally very efficient in delivering genetic material 2. Possible to target certain cell types 3. The virus usually stimulate the immune response and augments the vaccine specific response CONS More complex and expensive to produce than plasmid vaccines Immunity to the recombinant virus prevents repeated vaccinations with the same vaccine construct
22 Example of therapeutic HIV vaccineThe plasmids The recombinant Modified Vaccinia virus Ankara HIVIS: Example of therapeutic HIV vaccine
23 Prime Boost Strategy Immune Response Prime Boost 23Thailand the studies are happening for this prime boost strategy. We’ll also come back to this concept later on in the presentation. Prime Boost 23
24 Heterologous Prime-BoostMonths 1 2 3 6 9 12 Plasmid DNA Same antigen in another form: recombinant virus vaccine; protein; peptides etc Svårt ord – heterolog (olika) Prime priming av immunsvaret, boost – ökning eller extra skjuts. Denna strategianvänds för att dra nytta av fördelarna av de båda vaccinstrategierna. PÅ detta sätt så får man en fokusering av svaret mot sitt vaccinantigen – man primar med plasmid en “ren” immunisering som vanligtvis ger ett lågt men väldigt specifikt svar. När man sedan går in med boosten så ger man detta svar en rejäl skjuts och det blir rejäl skjuts på immunsvaret. Same antigen encoded by the plasmid and presented by the heterologous boost; Focuses the immune system on the vaccine antigen G. Karlsson-Hedestam
25 DNA DNA DNA MVA MVA Sacrifice Week 3 7 11 15 17Wahren B et al, 2007 DNA DNA DNA MVA MVA Sacrifice Week 3 7 11 15 17 25ug/plasmid injected intradermally using the Biojector. Plasmid injected as 2 entities (env/rev and gag/RT) 1ug rGM-CSF injected at the site of injection of envelope-encoding DNA Animals boosted with 107 pfu i.m. rMVA
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27 Thai HIV Vaccine Study Community-based studyNot specifically high-risk Moderate risk more likely predominated 31.2% Efficacy to prevent infection Two-tailed p value with 95% CI 1.1%-52.1% 51/8197 infected vaccinees 74/8198 infected placebo Rerks-Ngarm S, Pitisuttithum P et al, 2009
28 From HIVIS to TaMoVac Designation 2004 2006 2007 2009 2010 2011N (+placebo) 2004 2006 2007 2009 2010 2011 2014 HIVIS01/02/05 Stockholm phase 1 3xDNA 1st MVA Published 2nd MVA HIVIS03/ Dar es Salaam 40 (+20) phase 1/2 3xDNA + 1st MVA 3rd MVA Analysis ongoing TaMoVac I (Tz) Dar + Mbeya 108 (+12) phase 2 3xDNA 2xMVA rgp140/ GLA I: In press II: Manuscript TaMoVac I (Moz) Maputo 20 (+4) phase 1 HIVIS07 Stockholm 22 (+5) phase 1 3xDNA +/- EP 2xMVA +/-rgp140 Manuscript submitted TaMoVac II Dar+ Mbeya+ Maputo 180 +(18) phase 2 3xDNA +/- EP Addition of rgp140/GLA to MVA boost MVA: MVA-CMDR DNA: 7 plasmid DNA multigene/multisubtype vaccine EP: Electroporation
29 Thai HIV Vaccine Trial: Surprising findingsProtection correlated with Ab against V2 region Early, high protective immune response First 12 months post-vaccination cumulative vaccine efficacy was est. 60.5% (95 % CI 22–80) Efficacy declined quickly Hence: Maybe additional boost or other ↑immune response can ↑ efficacy.
30 Papilloma Virus Vaccines
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32 Papilloma virus gene function(From Fields Virology, 4th ed, Knipe & Howley, eds, Lippincott Williams & Wilkins, 2001, Table 66-1)
33 VACCINATION AIMS: VACCINATION APPROACHES:
34 Cervarix™, GlaxoSmithKline HPV 16, 18 Vaccines are based on virus-like particles (VLPs) derived from the non-infectious L1 outer capsid proteins (Fife, Wheeler et al. 2004). The vaccines are non-infectious. Nearly all participants in clinical trials of HPV vaccines have demonstrated seroconversion with antibody levels several-fold higher than those observed following natural infection (Harper, Franco et al. 2004; Sattler and Investigators. 2005; Skjeldestad and Committee 2005; Villa, Costa et al. 2005) Minimal antibody levels considered to provide protection against each of the HPV subtypes have not yet been established. Moreover, antibody levels are HPV type–specific and assay-specific, and it is not possible to compare antibody levels across or within trials. Cervarix™, GlaxoSmithKline HPV 16, 18 Gardasil®, Merck. HPV 6, 11, 16, 18 Harper, Franco et al. 2004; Sattler et al 2005; Skjeldestad and Committee 2005; Villa, Costa et al. 2005
35 Gardasil® protects against the HPV types 16, 18, 6, and 11. Three FDA-approved vaccines—Gardasil®, Gardasil-9®, and Cervarix®—prevent HPV infection and therefore guard against the major cause of cervical and ano-genital cancers and potentially head and neck cancer. Gardasil® protects against the HPV types 16, 18, 6, and 11. Gardasil-9® is approved for the prevention of cervical, vulvar, vaginal, and anal cancers caused by HPV types 16, 18, 31, 33, 45, 52, and 58, and for the prevention of genital warts caused by HPV types 6 or 11 Men between the ages of 9 and 26 may receive Gardasil-9® to protect their future partners and to protect themselves against anal cancer and potentially head and neck cancer, as well as genital warts. Cervarix is FDA approved for use in preventing the two strains of HPV that cause most cervical cancers, HPV 16 and 18.
36 Anti-tumor DNA vaccines based on the expression of HPV16 E6/E7 oncoproteinsC57Bl/6 mice immunized with gD/E6/E7 DNA and challenged with neoplastic TC-1 cells expressing HPV16 E6 & E7 Lasaro MO et al, 2005
37 First HPV DNA vaccines clinical trialsDNA vaccine encoding a signal sequence linked to E7 with abolished Rb binding site (E7detox) and fused to heat shock protein 70 (Sig/E7detox/Hsp70) - Phase I trials on HPV-16 positive patients with high-grade CIN lesions 2/3. Homologous DNA-prime-boost vaccination regimen of three vaccinations per patient, at three dose levels, 500, 1,000, and 3,000 ug. Regression in 3/9 pts (Dr Trimble, Johns Hopkins University). DNA vaccine encoding calreticulin (CRT) fused to E7detox using a PowderMed/ Pfizer proprietary gene gun device - Phase I in HPV-16 positive patients with stage 1B1 cervical cancer (Dr Alvarez, University of Alabama at Birmingham) Hung CF et al, DNA vaccines for cervical cancer, 2007
38 Therapeutic DNA-based HPV VaccinesA phase II clinical trial of TVGV-1 vaccine for patients with HPV-induced cervical pre-cancer (NCT ). A phase I/II trial of VGX-3100, a vaccine that targets HPV types 16 and 18, and INO-9012, a DNA construct that induces human interleukin 12 (IL-12), are being tested in patients with cervical cancer (NCT ). ADXS11-001, a vaccine against the E7 protein, which is made by HPV, is in phase I/II trials in patients with anal cancer (NCT ). There are two phase I clinical trials testing pNGVL4a/E7 (Detox)/HSP70 DNA vaccine in patients with HPV16+ cervical intraepithelial neoplasia. The first one will determine the best dose (NCT ) and the second one will be a combination with imiquimod, an innate immune activator (NCT ).
39 Reasons for failure of herpes candidate vaccinesPoorly controlled studies Insufficient dose Insufficient immunogenicity (Ab) No induction of CTL No induction of mucosal antibodies In case of recurrent disease, difficult to eradicate reservoir
40 Attempts to create a vaccine against Herpes simplexAuto-inoculation Live, deletion mutants (replication limited) Pox virus and adenovirus vectors for glycoproteins Inactivated whole virus Inactivated infected cell extracts Subunit glycoproteins Disabled Infectious Single Cycle (DISC) DNA plasmids Peptides Knowledge of natural immunity, including mucosal immunity Induction of CD4 and CD8 cell-mediated immunity (CMI) Local and systemic cellular immunity: importance of cytokines, particularly IFN, to recruit T and B cells, and up-regulate HLA Live or non-replicating virus to do this? HSV-1 glycoproteins gB, gC, gD, gE, gG, gH, gI
41 DNA-immunization Antigen Model AuthorsgB/Sindbis HSV-1, mouse Hariharen 1998 gB HSV-1, mouse Manicken 1995 gD/bupivacaine HSV-2, mouse.g.pig Bernstein 1999 tgB + gD HSV-2, mouse/g.pig McClements 1996/1997 gD + IL12 HSV-2, mouse Sin 1999 gD HSV-1, mouse Ghiasi 1995 gd/tgD BHV-1, cattle Van Drunnen 1998 ICP27 HSV-1, mouse Manicken 1995
42 Benefits of host immune response to CMVMaternal immunity ameliorates effects of intrauterine infection on fetus Premature neonates with maternal antibody are protected from postnatal infection Pre-transplant immunity protects organ allograft recipients from severe disease Passively administered antibody protects organ allograft recipients Protective efficacy demonstrated in murine and guinea pig models of (homologous strain) CMV infection using attenuated strains Experience with Towne vaccine in humans
43 Experimental CMV vaccinesLive attenuated Towne strain Recombinant of Towne with genes from virulent virus Subunit gB glycoprotein Subunit gH glycoprotein DNA plasmids gB in adenovirus vector Multiple genes in canarypox vector Prime boost with canarypox/subunit gB
44 //////////// hepe Hepatitis Viruses
45 Characteristics and results gB/MF59 adjuvant Phase II study completedVaccines Status Characteristics and results Ref. gB/MF59 adjuvant Phase II study completed Acceptable safety for further studies Evaluated in HCMV-seronegative women within 1 year after they had given birth Vaccine efficacy of 50% on the basis of infection rates per 100 person years [20] gB/pp65/IE1 alphavirus replicon trivalent vaccine Phase I study Favorable safety profile Evaluated in healthy, nonpregnant adults Elicits humoral and cellular immune responses Based on replication-deficient alphavirus technology [39] gB/pp65 bivalent DNA vaccine Ongoing Phase II study Well tolerated with no serious adverse events in a Phase I study HCMV-seropositive or -seronegative healthy adults in Phase I and HCT recipients in Phase II studies Higher frequencies of HCMV-specific pp65 and gB T cells compared with placebo [42,47] Towne ± rhIL-12 ± priming by DNA vaccine encoding pp65, IE1 and gB Phase I studies Favorable safety profile; no evidence for viral latency or viral shedding in recipients Evaluated in HCMV-seronegative healthy adults Augmentation of immunogenicity by inclusion of rhIL-12 or DNA vaccine in Phase I studies [52,53] Sung H & Schleiss MR, 2010
46 THANK YOU FOR YOUR ATTENTION!!!
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48 Immunization without NeedlesOral vaccines-polio, typhoid, cholera, rotovirus, nasal influenza Cutaneous vaccination: liquid-jet injection deliver antigen id, sq or im (Advantage-Langerhans cells in skin-requires lower dose of antigen. Disadvantage-blood transfer) Particle bombardment of the skin -epidermal powder administration Topical application to skin using adjuvants and or permeabilizing agents Problems with Needle Administration of Vaccines 750 million injection given annually Limitations -costs -needle phobia -accidental sticks -improper and unsafe use
49 New strategies for second generation vaccines based on cellular immunity.JA Berzofsky et al, 2004
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51 Bråve 2007 CD8+ T cell Somatic cell CTL Th1 cell APC CD4+ T cellDirect priming TCR IL-2 IFN-g TNF MHC I CD8 Cross priming Secreted or membrane bound antigen and/or apoptotic material Th1 cell APC TCR CD4 CD4+ T cell MHC II IL-4 IL-5 IL-10 B cell Plasma B cell Vaccine- encoded antigen Th2 cell Secretion of antibodies Bråve 2007
52 Benefits of host immune response to CMVMaternal immunity ameliorates effects of intrauterine infection on fetus Premature neonates with maternal antibody are protected from postnatal infection Pre-transplant immunity protects organ allograft recipients from severe disease Passively administered antibody protects organ allograft recipients Protective efficacy demonstrated in murine and guinea pig models of (homologous strain) CMV infection using attenuated strains Experience with Towne vaccine in humans
53 Experimental CMV vaccinesLive attenuated Towne strain Recombinant of Towne with genes from virulent virus Subunit gB glycoprotein Subunit gH glycoprotein DNA plasmids gB in adenovirus vector Multiple genes in canarypox vector Prime boost with canarypox/subunit gB
54 The cells of the body acts as vaccine factoriesPresentation of protein fragments on MHC class I molecules Secreted protein DNA protein translation mRNA transcription Poängen med genetiska vacciner är att kroppens celler själva producerar vaccinprotein. Cellerna tar upp plasmiden och börja producera proteinet som kodas I plasmiden. På detta sätt simulerar man en virusinfektion och man kan stimulera delar av immunförsvaret (det cellulära), som man inte gör när man vaccinerar med protein direkt. Muscle cell G. Karlsson-Hedestam, SMI 2006
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56 Vaccines that stimulate Killer T cells protect rhesus monkeys against SIV/SHIV infection:Barouch et al DNA + IL-2 Amara et al DNA + rMVA Shiver et al DNA/rAdV Rose et al rVSV Consistent reduction of virus load by fold after challenge with virus Note SIV challenge is times human infecting dose: Nairobi Sex Workers take >100 contacts before half are infected
57 + C AC A multigene-/multisubtype vaccine for East Africa A 30% 34% AD9% D 6% C 30% AC 34% CD ACD AD Tanzania p37 gag B p37 gag A gp160 env B gp160 env A gp160 env C rev B RTmut B + MVA-HIV CRF_AE gag A/pol A/env E Vi har valt att inkludera gener som kodar för flera delar av HIV – detta föra tt bredda svaret och inte förlika sig på svar mot endsast en komponent. Vi har valt att inkludera både interna struktirella delar av HIV .nukleokapsiden, ytproteinerna samt några av de enzymer HIV kodar för. Vi har designat vaccinet med målet att vaccinera I östra afrika och mer specifikt I Tanzania, vi har därför valt att inkludera gener från HIV av olika subtyper. Så här ser vårt vaccin ut, sju olika plasmider som kodar för de proteiner jag talade om. Vår rekombinanta virusvektor är som sagt modified vaccinia ankara och det kodar också för tre olika delar av HIV.
58 HIVIS vaccin i huden med elektroporering. Vaccinet produceras från DNA HIVs DNA gener direkt i kroppens celler. Total length of pulse-train: 0,27 seconds Time Voltage 2x450V 8x110V