Chapter 7 Final Discussion
7.2 Future work arising from this work
We now propose to consolidate the work completed in the initial phase of this project, reviewed in this thesis, by extending it out to other research projects including:
We plan to undertake transcriptonomic evaluation of PAXgene samples frozen at the Screening Visit/Days 0, 1, 7, 14, 28, 56, 57, and 63.
As HCV exists as distinct genotypes that are broadly segregated geographically, previous work in healthy volunteers has assessed the capacity of T cells generated by MVA boost encoding a subtype 1b immunogen to target genotypes 1a, 3a, and 4a. It was found that cross-reactive T cells responses between heterologous viral genotypes are readily generated but at a reduced magnitude [195]. We plan to assess these responses in the vaccinated HIV seropositive patients. Whether these responses observed in healthy volunteers are sufficient to provide protection remains unknown and will require efficacy studies in
mixed genotype populations. Further assessment of the significant T cell responses in Pool G in 2 of the 20 patients prior to administration of the vaccines will also be evaluated. This will be achieved by examining ‘mini-pools’ of the Pool G peptides in an effort to determine which peptide(s) are reactive.
HLA typing has been performed on all patients in the vaccine study. In the HCV003 study, the AdCh3NSmut / MVA-NSmut vaccine approach generated very high numbers of both CD4+ and CD8+ T cells, targeting multiple HCV antigens irrespective of host HLA background [195]. We plan to look at the magnitude and phenotype of HCV-specific CD8+ T cell populations identified by tetramer staining, before and after vaccination, in patients with the appropriate HLA class I alleles.
Several markers of T cell functionality (e.g. perforin and granzyme B expression) and the T cell phenotype (e.g. effector memory T cells) have been associated with HCV infection control. Therefore, we plan to use frozen PBMCs to evaluate exploratory immunology assays (including intracellular cytokine staining (ICS) phenotyping, viral suppression in vitro and proliferation). Included in this work, we plan to evaluate whether CD4+ or CD8+ T cells were the predominant producers of IFN-g. We also plan to evaluate for rates of multifunctional T cells by assessing production of IFN-g, IL-2 and TNF-a. Previous work in the HCV003 trial demonstrated the polyfunctionality of CD4+ and CD8+ cells peaked at week 18 and 22 of the trial respectively, following MVA vaccination [195]. We plan to compare our results with the healthy volunteers who received the same vaccine strategy in the PEACHI-04 study at the University of Oxford.
Similarly using frozen PBMCs and serum for innate immunological assays we plan to measure natural killer cell (NK) function, at specific time points including before, within 24 hours and 7 days after vaccination. The relationship between innate and adaptive immune responses to HCV vaccination will be explored.
One of the host factors that may determine the immunogenicity of AdCh3NSmut1 in our study population is the potential for neutralisation of the vaccine by pre-
existing adenovirus-specific antibodies. This hypothesis is based on the observed lower magnitude responses to encoded HIV proteins among recipients of Merck’s trivalent Ad5-vectored HIV-1 vaccine with pre-existing humoral immunity to Ad5 [251, 252]. We therefore considered the following possibilities in the design of the vaccine trial: 1) neutralisation of the AdCh3 vector by pre- existing AdCh3-specific antibodies; 2) neutralisation of the AdCh3 vector by pre- existing antibodies to human adenoviruses that could cross-react with AdCh3.
PEACHI consortium members have screened sera from 193 human subjects from Europe and the US for neutralising antibodies against a panel of human and chimpanzee adenoviruses: AdCh3-specific antibody positivity (titre >200) was observed in just over 10%, while 40% had Ad5-specific antibody titres >200 [190]. Furthermore, in another study, AdCh-specific neutralising antibodies were rarely detected among human subjects from the US and Thailand and prevalance was ≤10% in subjects from sub-Saharan Africa [253]. Colloca et al also found that pre-existing Ad5-specific antibodies did not cross-neutralise AdCh3 in mice that were vaccinated with a AdCh3-vectored HIV-1 immunogen: T cell responses to the encoded HIV-1 proteins were similar in Ad5-immune and control mice [190].
Taken together with data from phase I clinical trials showing strong T cell responses, following vaccination of healthy subjects with AdCh3NSmut, the risk of pre-existing humoral immunity to adenoviruses impairing vaccine responses in our study is considered to be low. However, to address this question definitely, serum samples from study participants were stored for analysis of neutralising activity against a panel of human and chimpanzee adenoviruses including AdCh3. These samples have now been shipped to our colleagues at ReiThera in Rome for further analysis.
We plan to further evaluate the whole blood stimulation assay with further analysis of IP-10, IFN-ɣ and interleukin 2 (IL-2) detections by ELISA in supernatants and the correlation of chemokine / cytokine secretion and respective mRNA cell levels.
Given, 2 of the patients who participated in the vaccine trial became HCV antibody positive at the last clinic visit, we plan to analyse Day 0 and Day 238 serum samples from patients with a spectrum of Hepatitis C antibody assays, to further understand the antibody response in these patients. As mentioned before, no patient had evidence of detectable HCV by RNA testing at any point in the study.
PEACHI investigators have also developed further vaccines using adenoviral and MVA vectors when the encoded HCV immunogen (NSmut) is fused to MHC class II invariant chain (Ii). We plan an evaluation of a vaccine strategy in healthy volunteers in Oxford initially. If safety data are favourable, we plan to expand this vaccine strategy to further populations including patients who have been successfully treated with IFN-free DAA regimens and patients with HIV infection.
References
1. Feinstone, S.M., et al., Transfusion-associated hepatitis not due to viral
hepatitis type A or B. N Engl J Med, 1975. 292(15): p. 767-70.
2. Bradley, D.W., et al., Posttransfusion non-A, non-B hepatitis in chimpanzees. Physicochemical evidence that the tubule-forming agent is a small,
enveloped virus. Gastroenterology, 1985. 88(3): p. 773-9.
3. Choo, Q.L., et al., Isolation of a cDNA clone derived from a blood-borne non-
A, non-B viral hepatitis genome. Science, 1989. 244(4902): p. 359-62.
4. Choo, Q.L., et al., Genetic organization and diversity of the hepatitis C virus.
Proc Natl Acad Sci U S A, 1991. 88(6): p. 2451-5.
5. Simmonds, P., The origin and evolution of hepatitis viruses in humans. J Gen Virol, 2001. 82(Pt 4): p. 693-712.
6. Simmonds, P., et al., Consensus proposals for a unified system of
nomenclature of hepatitis C virus genotypes. Hepatology, 2005. 42(4): p.
962-73.
7. Simmonds, P., The origin of hepatitis C virus. Curr Top Microbiol Immunol, 2013. 369: p. 1-15.
8. Ishak, K., et al., Histological grading and staging of chronic hepatitis. J Hepatol, 1995. 22(6): p. 696-9.
9. Westbrook, R.H. and G. Dusheiko, Natural history of hepatitis C. J Hepatol, 2014. 61(1 Suppl): p. S58-68.
10. Seeff, L.B., Natural history of chronic hepatitis C. Hepatology, 2002. 36(5 Suppl 1): p. S35-46.
11. Bjoro, K., et al., Hepatitis C infection in patients with primary
hypogammaglobulinemia after treatment with contaminated immune
globulin. N Engl J Med, 1994. 331(24): p. 1607-11.
12. Kenny-Walsh, E., Clinical outcomes after hepatitis C infection from
contaminated anti-D immune globulin. Irish Hepatology Research Group. N
Engl J Med, 1999. 340(16): p. 1228-33.
13. Wiese, M., et al., Low frequency of cirrhosis in a hepatitis C (genotype 1b) single-source outbreak in germany: a 20-year multicenter study.
Hepatology, 2000. 32(1): p. 91-6.
14. Thein, H.H., et al., Estimation of stage-specific fibrosis progression rates in chronic hepatitis C virus infection: a meta-analysis and meta-regression.
Hepatology, 2008. 48(2): p. 418-31.
15. Peters, M.G. and N.A. Terrault, Alcohol use and hepatitis C. Hepatology, 2002. 36(5 Suppl 1): p. S220-5.
16. Tsukuma, H., et al., Risk factors for hepatocellular carcinoma among
patients with chronic liver disease. N Engl J Med, 1993. 328(25): p. 1797-
801.
17. Bulteel, N., et al., Factors associated with spontaneous clearance of chronic hepatitis C virus infection. J Hepatol, 2016. 65(2): p. 266-72.
18. Negro, F., et al., Extrahepatic morbidity and mortality of chronic hepatitis C.
Gastroenterology, 2015. 149(6): p. 1345-60.
19. Foster, G.R., R.D. Goldin, and H.C. Thomas, Chronic hepatitis C virus infection causes a significant reduction in quality of life in the absence of
20. Okuda, M., et al., Differences in hypervariable region 1 quasispecies of hepatitis C virus in human serum, peripheral blood mononuclear cells, and liver. Hepatology, 1999. 29(1): p. 217-22.
21. Zignego, A.L., et al., Hepatitis C virus infection of mononuclear cells from
peripheral blood and liver infiltrates in chronically infected patients. J Med
Virol, 1995. 47(1): p. 58-64.
22. Zarski, J.P., et al., Characteristics of patients with dual infection by hepatitis B and C viruses. J Hepatol, 1998. 28(1): p. 27-33.
23. Vento, S., et al., Fulminant hepatitis associated with hepatitis A virus
superinfection in patients with chronic hepatitis C. N Engl J Med, 1998.
338(5): p. 286-90.
24. EASL Recommendations on Treatment of Hepatitis C 2016. J Hepatol, 2017.
66(1): p. 153-194.
25. Eyster, M.E., et al., Heterosexual co-transmission of hepatitis C virus (HCV)
and human immunodeficiency virus (HIV). Ann Intern Med, 1991. 115(10):
p. 764-8.
26. Soto, B., et al., Heterosexual transmission of hepatitis C virus and the possible role of coexistent human immunodeficiency virus infection in the
index case. A multicentre study of 423 pairings. J Intern Med, 1994. 236(5):
p. 515-9.
27. Lauer, G.M. and B.D. Walker, Hepatitis C virus infection. N Engl J Med, 2001.
345(1): p. 41-52.
28. Kamili, S., et al., Laboratory diagnostics for hepatitis C virus infection. Clin Infect Dis, 2012. 55 Suppl 1: p. S43-8.
29. EASL-ALEH Clinical Practice Guidelines: Non-invasive tests for evaluation of
liver disease severity and prognosis. J Hepatol, 2015. 63(1): p. 237-64.
30. Pawlotsky, J.M., Hepatitis C Virus Resistance to Direct-Acting Antiviral
Drugs in Interferon-Free Regimens. Gastroenterology, 2016. 151(1): p. 70-
86.
31. Fourati, S. and J.M. Pawlotsky, Virologic Tools for HCV Drug Resistance Testing. Viruses, 2015. 7(12): p. 6346-59.
32. Bruno, S., et al., Survival of patients with HCV cirrhosis and sustained
virologic response is similar to the general population. J Hepatol, 2016.
64(6): p. 1217-23.
33. van der Meer, A.J., et al., Association between sustained virological response and all-cause mortality among patients with chronic hepatitis C and
advanced hepatic fibrosis. Jama, 2012. 308(24): p. 2584-93.
34. Takahashi, K., et al., Plasmacytoid dendritic cells sense hepatitis C virus-
infected cells, produce interferon, and inhibit infection. Proc Natl Acad Sci U
S A, 2010. 107(16): p. 7431-6.
35. Wilkins, C. and M. Gale, Jr., Recognition of viruses by cytoplasmic sensors.
Curr Opin Immunol, 2010. 22(1): p. 41-7.
36. Kawai, T. and S. Akira, The roles of TLRs, RLRs and NLRs in pathogen
recognition. Int Immunol, 2009. 21(4): p. 317-37.
37. Protzer, U., M.K. Maini, and P.A. Knolle, Living in the liver: hepatic
infections. Nat Rev Immunol, 2012. 12(3): p. 201-13.
38. Heim, M.H., Innate immunity and HCV. J Hepatol, 2013. 58(3): p. 564-74. 39. Isaacs, A. and J. Lindenmann, Virus interference. I. The interferon. Proc R
40. Blatt, L.M., et al., The biologic activity and molecular characterization of a
novel synthetic interferon-alpha species, consensus interferon. J Interferon
Cytokine Res, 1996. 16(7): p. 489-99.
41. Diaz, M.O., S. Bohlander, and G. Allen, Nomenclature of the human
interferon genes. J Interferon Cytokine Res, 1996. 16(2): p. 179-80.
42. Roberts, R.M., et al., The evolution of the type I interferons. J Interferon Cytokine Res, 1998. 18(10): p. 805-16.
43. Grander, D., O. Sangfelt, and S. Erickson, How does interferon exert its cell
growth inhibitory effect? Eur J Haematol, 1997. 59(3): p. 129-35.
44. Biron, C.A., Interferons alpha and beta as immune regulators--a new look.
Immunity, 2001. 14(6): p. 661-4.
45. Vilcek, J., Novel interferons. Nat Immunol, 2003. 4(1): p. 8-9.
46. Spencer, T.E. and F.W. Bazer, Conceptus signals for establishment and
maintenance of pregnancy. Reprod Biol Endocrinol, 2004. 2: p. 49.
47. Pestka, S., C.D. Krause, and M.R. Walter, Interferons, interferon-like
cytokines, and their receptors. Immunol Rev, 2004. 202: p. 8-32.
48. Kotenko, S.V., et al., IFN-lambdas mediate antiviral protection through a
distinct class II cytokine receptor complex. Nat Immunol, 2003. 4(1): p. 69-
77.
49. Platanias, L.C., Mechanisms of type-I- and type-II-interferon-mediated
signalling. Nat Rev Immunol, 2005. 5(5): p. 375-86.
50. Stattermayer, A.F., et al., Polymorphisms of interferon-lambda4 and IL28B - effects on treatment response to interferon/ribavirin in patients with
chronic hepatitis C. Aliment Pharmacol Ther, 2013.
51. Ge, D., et al., Genetic variation in IL28B predicts hepatitis C treatment-
induced viral clearance. Nature, 2009. 461(7262): p. 399-401.
52. Stevenson, N.J., et al., Hepatitis C virus targets the interferon-alpha
JAK/STAT pathway by promoting proteasomal degradation in immune cells
and hepatocytes. FEBS Lett, 2013. 587(10): p. 1571-8.
53. Gerlach, J.T., et al., Recurrence of hepatitis C virus after loss of virus-specific
CD4(+) T-cell response in acute hepatitis C. Gastroenterology, 1999.
117(4): p. 933-41.
54. Lauer, G.M., et al., High resolution analysis of cellular immune responses in
resolved and persistent hepatitis C virus infection. Gastroenterology, 2004.
127(3): p. 924-36.
55. Lechner, F., et al., CD8+ T lymphocyte responses are induced during acute
hepatitis C virus infection but are not sustained. Eur J Immunol, 2000.
30(9): p. 2479-87.
56. Diepolder, H.M., et al., Possible mechanism involving T-lymphocyte response to non-structural protein 3 in viral clearance in acute hepatitis C virus
infection. Lancet, 1995. 346(8981): p. 1006-7.
57. Thimme, R., et al., Determinants of viral clearance and persistence during
acute hepatitis C virus infection. J Exp Med, 2001. 194(10): p. 1395-406.
58. Takaki, A., et al., Cellular immune responses persist and humoral responses decrease two decades after recovery from a single-source outbreak of hepatitis C. Nat Med, 2000. 6(5): p. 578-82.
59. Bassett, S.E., et al., Protective immune response to hepatitis C virus in chimpanzees rechallenged following clearance of primary infection.
60. Bowen, D.G. and C.M. Walker, Adaptive immune responses in acute and
chronic hepatitis C virus infection. Nature, 2005. 436(7053): p. 946-52.
61. Rajoriya, N., et al., Gamma Delta T-lymphocytes in Hepatitis C and Chronic
Liver Disease. Front Immunol, 2014. 5: p. 400.
62. Klenerman, P. and R. Thimme, T cell responses in hepatitis C: the good, the
bad and the unconventional. Gut, 2012. 61(8): p. 1226-34.
63. Saito, H., et al., Complete primary structure of a heterodimeric T-cell
receptor deduced from cDNA sequences. Nature, 1984. 309(5971): p. 757-
62.
64. Chien, Y.H. and M. Bonneville, Gamma delta T cell receptors. Cell Mol Life Sci, 2006. 63(18): p. 2089-94.
65. Norris, S., et al., Natural T cells in the human liver: cytotoxic lymphocytes with dual T cell and natural killer cell phenotype and function are
phenotypically heterogenous and include Valpha24-JalphaQ and
gammadelta T cell receptor bearing cells. Hum Immunol, 1999. 60(1): p.
20-31.
66. Triebel, F. and T. Hercend, Subpopulations of human peripheral T gamma
delta lymphocytes. Immunol Today, 1989. 10(6): p. 186-8.
67. Agrati, C., et al., Gammadelta T cell activation by chronic HIV infection may contribute to intrahepatic vdelta1 compartmentalization and hepatitis C virus disease progression independent of highly active antiretroviral
therapy. AIDS Res Hum Retroviruses, 2001. 17(14): p. 1357-63.
68. Jung, M.K. and E.C. Shin, Regulatory T Cells in Hepatitis B and C Virus
Infections. Immune Netw, 2016. 16(6): p. 330-336.
69. Shevach, E.M., et al., The lifestyle of naturally occurring CD4+ CD25+
Foxp3+ regulatory T cells. Immunol Rev, 2006. 212: p. 60-73.
70. Zhao, D.M., et al., Activated CD4+CD25+ T cells selectively kill B
lymphocytes. Blood, 2006. 107(10): p. 3925-32.
71. Ghiringhelli, F., et al., The role of regulatory T cells in the control of natural
killer cells: relevance during tumor progression. Immunol Rev, 2006. 214:
p. 229-38.
72. Mahnke, K. and A.H. Enk, Dendritic cells: key cells for the induction of
regulatory T cells? Curr Top Microbiol Immunol, 2005. 293: p. 133-50.
73. Boettler, T., et al., T cells with a CD4+CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T cells during chronic hepatitis C virus infection. J Virol, 2005. 79(12): p. 7860-7.
74. Claassen, M.A., et al., Abundant numbers of regulatory T cells localize to the liver of chronic hepatitis C infected patients and limit the extent of fibrosis. J Hepatol, 2010. 52(3): p. 315-21.
75. Alter, M.J., Epidemiology of viral hepatitis and HIV co-infection. J Hepatol, 2006. 44(1 Suppl): p. S6-9.
76. Troisi, C.L., et al., A multicenter study of viral hepatitis in a United States
hemophilic population. Blood, 1993. 81(2): p. 412-8.
77. Gottlieb, M.S., et al., Pneumocystis carinii pneumonia and mucosal candidiasis in previously healthy homosexual men: evidence of a new
acquired cellular immunodeficiency. N Engl J Med, 1981. 305(24): p. 1425-
78. Masur, H., et al., Opportunistic infection in previously healthy women. Initial
manifestations of a community-acquired cellular immunodeficiency. Ann
Intern Med, 1982. 97(4): p. 533-9.
79. Barre-Sinoussi, F., et al., Isolation of a T-lymphotropic retrovirus from a
patient at risk for acquired immune deficiency syndrome (AIDS). Science,
1983. 220(4599): p. 868-71.
80. Gallo, R.C., et al., Frequent detection and isolation of cytopathic retroviruses
(HTLV-III) from patients with AIDS and at risk for AIDS. Science, 1984.
224(4648): p. 500-3.
81. Weiss, R.A., Gulliver's travels in HIVland. Nature, 2001. 410(6831): p. 963- 7.
82. Wawer, M.J., et al., Rates of HIV-1 transmission per coital act, by stage of
HIV-1 infection, in Rakai, Uganda. J Infect Dis, 2005. 191(9): p. 1403-9.
83. Buchbinder, S.P., et al., Sexual risk, nitrite inhalant use, and lack of
circumcision associated with HIV seroconversion in men who have sex with
men in the United States. J Acquir Immune Defic Syndr, 2005. 39(1): p. 82-
9.
84. Gray, R., et al., The effectiveness of male circumcision for HIV prevention
and effects on risk behaviors in a posttrial follow-up study. Aids, 2012.
26(5): p. 609-15.
85. Dorak, M.T., et al., Transmission of HIV-1 and HLA-B allele-sharing within
serodiscordant heterosexual Zambian couples. Lancet, 2004. 363(9427): p.
2137-9.
86. Mellors, J.W., et al., Plasma viral load and CD4+ lymphocytes as prognostic
markers of HIV-1 infection. Ann Intern Med, 1997. 126(12): p. 946-54.
87. Phillips, A.N., et al., Immunodeficiency and the risk of death in HIV infection.
Jama, 1992. 268(19): p. 2662-6.
88. Adler, M.W., ABC of Aids: Development of the epidemic. Bmj, 2001.
322(7296): p. 1226-9.
89. Cohen, M.S., et al., The spread, treatment, and prevention of HIV-1:
evolution of a global pandemic. J Clin Invest, 2008. 118(4): p. 1244-54.
90. Giese, C., et al., Injection of new psychoactive substance snow blow associated with recently acquired HIV infections among homeless people
who inject drugs in Dublin, Ireland, 2015. Euro Surveill, 2015. 20(40).
91. Tuite, H., et al., Patients Accessing Ambulatory Care for HIV-infection:
Epidemiology and Prevalence Assessment. Ir Med J, 2015. 108(7): p. 199-
202.
92. Samji, H., et al., Closing the gap: increases in life expectancy among treated
HIV-positive individuals in the United States and Canada. PLoS One, 2013.
8(12): p. e81355.
93. Gunthard, H.F., et al., Antiretroviral Drugs for Treatment and Prevention of HIV Infection in Adults: 2016 Recommendations of the International
Antiviral Society-USA Panel. Jama, 2016. 316(2): p. 191-210.
94. Platt, L., et al., Prevalence and burden of HCV co-infection in people living
with HIV: a global systematic review and meta-analysis. Lancet Infect Dis,
2016. 16(7): p. 797-808.
95. Nelson, P.K., et al., Global epidemiology of hepatitis B and hepatitis C in
people who inject drugs: results of systematic reviews. Lancet, 2011.
96. van der Helm, J.J., et al., The hepatitis C epidemic among HIV-positive MSM:
incidence estimates from 1990 to 2007. Aids, 2011. 25(8): p. 1083-91.
97. Thomson, E.C., et al., Predicting spontaneous clearance of acute hepatitis C
virus in a large cohort of HIV-1-infected men. Gut, 2011. 60(6): p. 837-45.
98. Wandeler, G., et al., Hepatitis C virus infections in the Swiss HIV Cohort
Study: a rapidly evolving epidemic. Clin Infect Dis, 2012. 55(10): p. 1408-
16.
99. McFaul, K., et al., Acute hepatitis C infection in HIV-negative men who have