1. URRMA R&D 1 LESSONS TO LEARN AFTER 30 YEARS OF HIV Medical University Plovdiv March 20th-21rst 2015 Prof. J C Chermann Jeanclaude.chermann@gmail.com +33620102343

2. Classification ONCORNAVIRUS LENTIVIRUS SPUMAVIRUS (FOAMY) 2

3. Definition RNA VIRUS Need an intermediary DNA to replicate Reverse transription made throught a reverse transcriptase Envelopped Virus Budd at the cell surface Density 1.16-1.18 in sucrose gradient 3

4. MORPHOLOGY A type Particule ( without known role) B type particule mainly Mammary Tumor Virus (MTV) C type virus mainly leukemia virus (MLV and HTLV1) D type lentivirus (Visna,Caev, Eiav, Fiv Siv,Hiv) 4

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12. THE REVERSE TRANSCRIPTASE The RT is composed by 3 enzymes: 1) the reverse transcriptase (RT) to copy the RNA in DNA 2) The Rnase H: to eliminate the RNA in the Hybrid RNA-DNA ( remain Ss DNA or complementary DNA) 3)a DNA polymerase to copy the DNA to Ds DNA Ready to integrate into the cellular genome, by an integrase 12

13. REVERSE TRANCRIPTASE The RT is a jumping enzyme starting the copy at the 5’ end and jump to the 3’end to continue the copy 13

14. VIRAL RNA Single strand RNA with a pseudo match of binding at the 5’end (two 5’end bind together). At this site bind the primer for the reverse transcriptase ( the primer is t- RNA lys.) Looks like a rabitt hear. Only RNA from the same virus will match 14

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17. THE HUMAN RETROVIRUSES 17

18. ORIGIN OF THE HUMAN RETROVIROLOGY With the following results for the SVCP The discovery of the Reverse Transcriptase with the birth of the Molecular Biology. The discovery of the oncogens with the transforming retroviruses (defective retroviruses) 18

19. HUMAN Retroviruses HTLV 1 and HTLV 2 Human T leukemia 1 or 2 ATLV:Adult T Leukemia Disease: Leukemia,myelopathy Found in Japan, Carribean island and Zaire 19

20. HTLV GENOME Beside the structural genes (gag, pol, env.) a new regulatory gene appears: the tax a transactivator gene. The product of this gene can act on different part of the cellular genome. With a result of a new mechanism of tumorisation 20

21. HUMAN Retroviruses Human Immunodeficiency virus HIV 1 and HIV 2 HIV 1 is the causal agent of AIDS and Associated Diseases. HIV 2 restricted in West Africa is also responsible for AIDS 21

22. URRMA R&D 22  Structure of HIV

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24. URRMA R&D 24   Genomic variability of proviral DNA  Mainly at the level of the ENV gene.  Also for the GAG and the POL genes.

25. URRMA R&D 25   Biological variability of HIV  Slow/low – fast/high replication. Ability to grow in T cell lines.  Low or high cytopathogenicity.  -CD4 affinity.  -V3 neutralizing antibodies (PNP).  -Cell tropism: - CD4 lymphotropism. - Macrophage tropism. - Dual tropism.

26. URRMA R&D 26   Variability at the level of the envelope glycoproteins: the V3 loop

27. The V3 loop variability URRMA R&D 27 Clade BClade D

28. URRMA R&D 28  Entry of HIV FIXATIONFUSION ENTRY

29. URRMA R&D 29   Another way to the CD4 for HIV entry into susceptible cells.  Replication of various strains – HIV in presence of monoclonal antibodies to the CD4 or soluble CD4. » Virol 1991,181,165-171 » J.Virol 1991,65:2102.2107.  Infection of CD4 negative cells. » Cancer letters:1992,63:23-31.

30. URRMA R&D 30  Coreceptor mechanism

31. URRMA R&D 31   Highly cytopathic virus HIV-NDK.  Zairian isolate, clade D.  10000 more cytopathic than the prototype.  Easy transmission.  Short incubation period.

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37. URRMA R&D 37   Isolation and characterization of HIV macrophage tropic strain:  From cerebrospinal fluid of an acute HIV infected patient presenting a dementia without immunodeficiency.

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42. URRMA R&D 42   Isolation and characterization of HIV macrophage tropic strain:  T lymphotropic virus can infect the macrophage without integration into the host genome. - Virology 1992,190:124-33  HIV for entry to the macrophage use several domains of the CD4. - Res Virol 1993,144: 21-26.  HIV binds to CCR5 coreceptor.

43. URRMA R&D 43 Characteristics of HIV-1 par: a macrophage tropic retrovirus.  Fully replicates - in monocyte-macrophage, - in cord blood lymphocytes, - in bone marrow progenitors.  Does not grow - in PBL. - in continous cell lines (H9,CEM,U937). » Schmidtmayerova et al.,Virology,1992,1990:124-133. » Schmidtmayerova et al.,Res.Virol.,Inst.Pasteur,1993,144:21-26.

44. URRMA R&D 44  Infected intestinal cells

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47. URRMA R&D 47  Infected intestinal cells

48. URRMA R&D 48  Infected intestinal cells

49. URRMA R&D 49   Intestinal cell can be infected by some HIV isolates:  Then malabsorption and « clean » diarrhea. » Proc.Natl.Acad Sci Usa 1991,88:9297-930 » J.Virol.1992,66:580-585 » J.AIDS 1992,5:993-1000   The receptor for the virus is the galactosyl - Ceramide. » J.Virol.1992,66:580-585

50. URRMA R&D 50  Bone marrow Progenitors infection

51. URRMA R&D 51  The CD34 cell can be infected by HIV and more often by HIV2 that infection is followed by  a lymphopenia  an anemia  a granulocytopenia  a trombocytopenia » AIDS Res.Hum.Retr.1992,8:711-718 » J.AIDS 1992,5:1148 – 1157 » AIDS 1992,6:943-948

52. URRMA R&D 52  Summary of HIV cellular targets HIV Anemia Lymphopenia Thrombocytopenia AIDS Dementia Pneumopathie …. intestinal cell Malabsorption ↓ Weight loss Hypersecretion ↓ Diarrhoea Progenitor CD34 Lymphocyte CD4 Macrophage Bone marrow

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54. URRMA R&D 54 THE FAILURE OF THE CLASSICAL WAY FOR THE VACCINE THE VARIABILITY OF HIV THE NEED OF AN HUMORAL AND MUCOSAL PROTECTION

55. URRMA R&D 55  Cellular and viral HIV antigens.

56. URRMA R&D 56  R7V epitope from the beta-2-microglobulin R T P K I Q V Arg Thr Pro Lys ILeu Gln Val R7V  2-m  -2-mR7V

57. URRMA R&D 57  Progressor definition  Regular CD4 cells decline  High plasmatic viral load  Virus isolation from CD4 cells  Non-progressor definition  HIV infected more than 10 years  Normal CD4 cells count  No disease

58. URRMA R&D 58  Retrospective study (SAU CH) -1991-11 years of a non-progressor non-treated patient

59. URRMA R&D 59  Retrospective study (TRA LA) -1983-3 years of a treated progressor patient.

60. URRMA R&D 60 RESULTS OF A STUDY ON AMERICAN SAMPLES

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65. URRMA R&D 65  Anti-R7V TM ELISA kit Clearly the presence of R7V antibodies correlates with a protection: In Infected patients with a survival time of more than 10 years (range 20 to 10), treated or not: more than 50% have protective antibodies 88.5% of HIV+ patients with anti-R7V antibodies survive without treatment more than 10 years ( range 10 to 20 years) 69.2% are protected without treatment more than 5 years of follow-up ( 5 to 10 years)

66. URRMA R&D 66  Results on anti-R7V antibodies

67. URRMA R&D 67  Neutralization of different groups of HIV-1 by purified human anti-R7V Abs 0 20 40 60 80 100 % NEUTRALIZATION UG92029 (A) RW92009 (A) BR92021 (B) BR93025 (C) UG92035 (D) BR93029 (F) BCF03 BCF 06 YBF30 LAV NDK X4 X4R5 BIOTYPE SI NSI _ R5 _ __ X4 GROUP MGROUP OGP NTCLA CO-RECEPTOR SI NSI

68. URRMA R&D 68  Correlation Between Presence of Anti-R7V Abs and Non Progression 1.Neutralizing antibodies neutralize laboratory strains and primary isolates. 2.Neutralizing antibodies directly neutralize the virus of the patient. 3.Dissociation of antigen (virus) antibody complex present in protected patients confirms the binding of antibodies with the virus 4.Sequential samples from non progressor or long term survivors show that only non-progressor patients have anti-R7V antibodies during all the course of the infection.

69.  Anti-R7V antibodies URRMA R&D 69  Are mainly IgG1 and/or IgG3.  Are present in non progressor patients during all the course of HIV infection and are maintained after treatment except with a combination for Epivir-Zerit for some patients.  Are neutralizing, immunoprecipitating and inducing viral lysis with complement even for HIV strains resistant to antiviral drugs (AZT, ddI, ddi/ddC) They can then be used as therapeutic antibodies for the patients in failure of combined antiviral therapy

70. R7V: a new immunogen for a vaccinal concept URRMA R&D 70

71. URRMA R&D 71  General Conclusions - The anti-R7V ELISA kit: an innovative tool for the clinicians to initiate or not a therapy for HIV-infected patients. Enable the physicians to save 25% of HIV drug and treatment costs in addition to sparing R7V+ patients the toxic effect of current combination therapies. - The anti-R7V therapeutic antibodies: for patients in failure of combined antiviral therapy antibody. - The R7V epitope: a new vaccinal way to induce humoral and mucosal protection.

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