Immune protection against dengue infection. Vaccine performance

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1 Immune protection against dengue infection. Vaccine performance SCOTT B HALSTEAD MD Department of Preventive Medicine Uniformed Services University of the Health Sciences

2 TOPICS Current dengue vaccines: development status The newly licensed tetravalent dengue live-attenuated vaccine is poorly effective and not safe. What can we learn about dengue viral vaccine protective immunity from Dengvaxia?

3 Vaccines

4 NIH National Institute of Allergy and Infectious Diseases, National Institutes of Health. USA

5 Molecular Attenuation / D4 Chimera Steve Whitehead, NIAID / Anna Durbin Johns Hopkins * * DEN4 5 C prm E NS1 NS2A NS2B NS3 NS4A NS4B NS5 3 DEN2/4 5 C prm E NS1 NS2A NS2B NS3 NS4A NS4B NS5 3 DEN1 5 C prm E NS1 NS2A NS2B NS3 NS4A NS4B NS5 3 DEN3 5 C prm E NS1 NS2A NS2B NS3 NS4A NS4B NS5 3 DEN4 * * DEN1 DEN2 DEN3 * = mutation restricts replication in liver cells * = increases growth in Vero cell, decreases infectivity for mosquitoes D30 D30 D30 D30

6 NIH VACCINE: Development strategy,humans Produce and test in human volunteers DEN 1 30, DEN 2 30, DEN 3 30, DEN Produce and test in human volunteers DEN1/4 30, DEN 2/4 30, DEN 3/4 30. Test monovalent and tetravalent candidates in human volunteers. Challenge vaccinated subjects with live attenuated dengue viruses.

7 NIH VACCINE: progress to date Individual DENV 1 30, DENV 2/4 30, DENV 3 30, DENV 4 30, % seroconversion in susceptible US adults. Combined vaccine resulted in >90% tetravalent seroconversion with acceptable reactogenicity. Complete protection against live DENV 2 challenge at 6 months.

8 NIH VACCINE: progress to date Antibody and T cell responses to vaccine closely resemble attributes of antibodies and T cells after natural primary and secondary dengue infections. Tetravalent vaccine licensed to Butantan, Phase 3 trials in progress.

9 TAKEDA

10 TAKEDA/Inviragen/CDC DENV 2 attenuated by 53 serial passages in primary dog kidney cells (DENV PDK 53 Mahidol vaccine). DENV 1, 3, 4 vaccines are chimeras based upon non-structural region of DENV 2 PDK 53 vaccine. DENV 1, 3, 4 structural regions are each derived from independently attenuated viruses (Mahidol vaccine).

11 TAKEDA VACCINE progress to date Tetravalent vaccine was immunogenic and protected rhesus monkeys after two doses. Phase 1/2 clinical trials in U.S. and Colombia. Phase 3 in progress in SE Asian and American dengue-endemic countries.

12 SANOFIPASTEUR

13 ChimeriVax TM technology YF 17D live vector Tom Monath & Farshad Guirakhoo, Acambis / Aventist Pasteur Yellow fever 17D genome cloned as cdna 5 C prm prm E E Non-structural Nonstructural genes genes 3 prm E prm E Exchange coat protein genes of dengue 1,2,3 and 4 (wild-type) 5 C Non-structural genes 3 Chimeric cdna > transcribe to RNA 5 3 Transfect mrna Grow virus in cell culture Envelope is heterologous virus containing immunizing antigens RNA replicative engine is YF 17D

14 Clinical Study CYD VACCINE (Yrs. 1-2) Phase IIb/III clinical trials /14: Subjects (2/3 vaccinated) Protective efficacy References Thailand % Sabcharoen et al Lancet Nov 2012 SE Asia 10, % Capeding et al Lancet Oct 2014 Americas 20, % Villar et al NEJM Jan 2015 Total 35, %

15 During 5 years post-3 rd dose of Dengvaxia 295 vaccinated children were hospitalized for a dengue infection* All ages, all 5 yrs: 295/20439 = 1.44%; Vaccinated as 2 5 y.o., 5th year = 2.8%* *Background Paper on Dengue Vaccine. SAGE document April 2016

16 What happened?

17 Remember Vaccinated populations in dengueendemic countries are mixtures: Seronegatives Monotypic dengue immunes Multitypic dengue immunes And in some places past infections with JE or YF or WN or Zika.

18 CYD VACCINE (1 yr post 3 rd dose) Vaccine efficacy in seronegatives Clinical Vaccine Control Study cases/at risk cases/at risk VE p SE Asia 23/424 18/ ( ) 0.16 Americas 9/258 9/ ( ) 0.24 Combined 32/682 27/ ( ) 0.08 (Hadinegoro et al NEJM 2015)

19 CYD VACCINE (1 yr post 3 rd dose) Vaccine efficacy in seropositives Clinical Vaccine Control Study cases/at risk cases/at risk VE p SE Asia 18/901 34/ ( ) <0.001 Americas 8/ / ( ) <0.001 Combined 26/ / ( ) <0.001 (Hadinegoro et al NEJM 2015)

20 Immune response in 95 children to Dengvaxia - prevalence of dengue neutralizing antibodies 28 days post 3 rd dose. (Sabchareon et al Lancet 2012) DENV 1 DENV 2 DENV 3 DENV 4 GMT %Aby 95% 99% 100% 98%

21 Vaccinated seronegatives circulate poorly protective antibodies - ADE a specific risk Intrinsic risk to DENV ADE is governed by age. Relevant data. Age specific hospitalization rates from 1981 DENV 2 epidemic in Cuba 3 years following nationwide virgin-soil DENV 1 epidemic (44% seroprevalence)

22 DENV antibodies in vaccinated seronegatives most closely resemble dengue neutralizing antibodies at birth in highly endemic countries. (mothers with 2 or more DENV infections) Example of maternal antibodies: DENV 1 DENV 2 DENV 3 DENV 4 PRNT

23 DSS in a 6 month-old infants with hepatomegaly. Vietnam

24 INFANT DHF/DSS CHILDREN'S HOSPITAL, BANGKOK, THAILAND AGE (months)

25

26 The same antibodies that protect, enhance at lower concentrations.

27 Passive antibodies enhance dengue 3 5x more efficiently than secondary DENV infections Primary Secondary 100/ /1000 (1) (2) 1) Halstead SB Togaviruses. Acad Press, ) Halstead SB. Adv. Virus Research, 2003

28 In vitro ADE model % Infection without serum Power Ratio of % infection at PENT/ Control Control PENT Peak enhancement titer = titer at maximal % infection for the serum tested Modified from Kliks et al, Am. J.Trop. Med Hyg 1989

29 NEW PARADIGM EXTRINSIC ADE increase in number of infected monocytes/macrophages. 3 X increase INTRINSIC ADE increase in virus production per infected cell X increase

30 Increased viral production with ADE in Mature DC >2-log 2.5x Serum dilution factor Marovich, M. personal communication

31 INTRINSIC ADE (iade) Observed with wide taxa of organisms capable of replicating in monocytes/macrophages. Requires attachment of non-protective enhancing IgG antibodies. Enhancing immune complex ligates Fc receptor which signals innate immune responses via tail.

32 ADE: The two pathways following Fc receptor ligation: 1) suppress type I IFN; 2) produce IL-10. (Ubol S Clin Vacc Imm 17:1829, 2010

33 Hospitalizations for breakthrough dengue infections in vaccinees Rates are higher than those for natural secondary dengue infections.

34 Hospitalization rate in vaccinated 2 5 y.o. seronegative children CYD VACCINE (2 yrs post 3rd dose) 20 hospitalizations in 2029 vaccinated children; 2 in 1005 placebo controls. Seronegatives (54.2%)* = 1100 DENV infection rate = 16%* DENV infected = 176 Hosp/DENV infected 20/176 = 11.4% Data from Coudeville Vaccine, 2016 and Halstead et al Vaccine, 2016

35 Hospitalization rate in 2 5 y.o. placebos (secondary DENV infections) Placebo group = 1005 % monotypic DENV immunes = 37.8%* Monotypic immunes = 379 DENV infections, 16%* x 379 = 61 Hospitalized = 2 Hospitalized/2 nd DENV inf. 2/61 = 3.3% Data from Coudeville et al Vaccine, 2016 And Halstead et al Vaccine, 2016.

36 Dengue hospitalization rates in vaccinated vs secondary DENV infection. DENV infection of vaccinated seronegatives is 3.5 times more likely to result in hospitalization than DENV-infected monotypic-immunes (placebo group)!

37 Sanofi says it is safe and effective to give Dengvaxia to >/= 9 year-olds. Is it?

38 CYD VACCINE (2 yrs. post 3 rd dose) Disease enhancement in 2-5 y.o. hospitalization phase 2 years CYD Age Vacc Hosp Contr Hosp Relative Risk = 5.0

39 Age at hospitalization of dengue-infected vaccinated children, 2 years post-3 rd dose

40 Dengue hospitalization rates/ 10,000 by age, 1981 Cuba GUZMAN MG et al. Int J Infect Dis 6:18, 2002

41 CAPILLARY FRAGILITY Gamble J et al. Biochem Soc Med Res Soc 98:211-6, 2000.

42 Furthermore, in SE Asia due to increasing solid immunity, hospitalization rate decreases after age 9.

43 ADMISSIONS FOR DENGUE INFECTION, QUEEN SIRIKIT NATIONAL INSTITUTE OF CHILD HEALTH, BANGKOK, THAILAND Combined

44 Age at dengue hospitalization 2 yrs post 3 rd dose of placebo Hadinegoro et al NEJM 2015

45 Why doesn t Dengvaxia protect seronegatives? Dengvaxia presents dengue envelope domain to immune system.

46 YF 17D raises antibodies to quaternanry epitopes on virion One dose results in circulation of neutralizing antibodies for a lifetime. Neutralizing antibodies long thought to mediate protection. Vratskikh et al have shown that YF 17D antibodies directed at E do not neutralize YFV. (PLoS Pathog 2013) Instead, neutralization is mediated by antibodies to quaternary epitopes on the complete virion.

47 Does Dengvaxia raise antibodies to quaternary epitopes?? Antibodies directed to quaternary epitopes to only DENV 4 in vaccinated seronegatives were observed (7/11). (Henein et al JID 2017) Antibodies directed to quaternary epitopes to DENV 1 3 in vaccinated seronegatives were rare (2/11). Protection against DENV 4 = ~ 80%

48 How is the Dengaxia virion structured?

49 ChimeriVax TM technology YF 17D live vector Tom Monath & Farshad Guirakhoo, Acambis / Aventist Pasteur Yellow fever 17D genome cloned as cdna 5 C prm prm E E Non-structural Nonstructural genes genes 3 prm E prm E Exchange coat protein genes of dengue 1,2,3 and 4 (wild-type) 5 C Non-structural genes 3 Chimeric cdna > transcribe to RNA 5 3 Transfect mrna Grow virus in cell culture Envelope is heterologous virus containing immunizing antigens RNA replicative engine is YF 17D

50 How does structure affect DENV T cell responses?

51 Dengue CD 4+ and CD 8+ T cells are directed at non-structural proteins HLA/dengue resistance studies have shown that human CD 8+T cells protect against severe secondary dengue infections. Human CD4+ and CD8+ T cell responses to the NIH vaccine are identical to responses following natural DENV infection.

52 Dengue T cell papers Weiskopf D et al PNAS 2013 Weiskopf D et al J Virol 2014 Weiskopf D et al PNAS 2015 Weiskopf D et al JID 2015 Weiskopf D et al JID 2016 Nivarthi UK et al J Virol 2016 Ngono A et al E Biomed 2016 Angelo MA et al J Virol 2017 Weiskopf D et al Front Immunol 2014 Weiskopf D et al Front Immunol 2016 Tian et al Front Immunol 2016

53 Epitope density in the DENV polyprotein # of donors responded % 7% 8% 6% 1% 3% 31% 3% 15% 22% relative response per antigen NS3, NS4B and NS5 are dominant CD8 + T cell targets 53

54 C, NS3 and NS5 antigens are dominant for CD4 responses A total of 363 epitopes recognized in at least two or more donors were identified Total=363 C M E NS1 NS2A NS2B NS3 NS4A NS4B NS5 magnitude of response [SFC/ 10 6 PBMC] % 54% C M E NS1 NS2A NS2B NS3 NS4A NS4B NS5 54% of the CD4 epitopes were derived from NS proteins 46% of them were derived from structural proteins.

55 Serotype-specific immunodominance T cell reactivity against all four serotypes is detected in the Nicaragua endemic area. Serotype specific differences in immuno-dominant protein patterns as a function of the serotype Also in Sri Lanka DENV3 specific responses are mostly dominated by responses against the structural proteins 55

56 Lessons learned from natural infection 1. Protein location of T cell targets. Responses against non-structural proteins are dominant. Immune dominance is influenced by the serotype. 2. HLA restriction of T cell targets.

57 Conclusions I. Different immunodominance pattern for Class I and Class II responses C is the main target for Class II Implications for mechanism of T cell help (Env is not a dominant target) CD4 Megapool allows study responses in small samples irrespective of HLA restriction and serotypes

58 Conclusions II. NIH vaccine (LAV) induces CD4 T cells responses NIH vaccine responses are higher than those observed in Natural Infection (NI). Immunodominance for CD4/NIH vaccine Differs from CD8 Resembles NI for CD4 Similar pattern of HLA dominance CD4 epitopes targets in DENV 2 LAV are comparable to natural infection NIH vaccine immunization induces balanced serotype responses 58

59 What has the inability of Dengvaxia to protect seronegatives taught us? Most likely explanation is absence of a fully competent CD4+ and CD8+ T cell response. T cell immunity seems essential to long lasting protection by viral vaccines. T cell immunity requires presentation of whole, replicating virus.

60 Successful viral vaccines (whole viruses, induce T cell immunity) Measles Mumps Japanese encephalitis (SA ) Rubella Smallpox Varicella Yellow fever

61 Killed viral vaccines raise protective antibodies. Do anamnestic antibody responses protect secondary organs? Japanese encephalitis Polio Tick-borne encephalitis

62 Think about it! Thank you