Immune Responses Induced by Immunization with HIV-1 DNA followed by HIV-Modified Vaccinia Virus Ankara with or without Recombinant gp 140 in Healthy Tanzanian Individuals THESIS FOR DOCTORAL DEGREE ( Ph . D . )

A vaccine against HIV is widely considered the most effective and sustainable way of preventing new infections. We previously conducted a phase I/II clinical trial using multi-clade, multigene HIV-DNA priming and boosting with the recombinant modified vaccinia Ankara (HIV-MVA) virus among healthy Tanzanian volunteers (HIVIS03 trial). The HIV-DNA vaccine contained seven plasmids expressing HIV-1 gp160 subtypes A, B, C, Rev B, Gag A, B and RTmut B and the recombinant HIV-MVA vaccine expressed CRF01_AE HIV-1 envelope (Env) subtype E and Gag-Pol subtype A. Sixty HIV-uninfected volunteers were randomized into three groups of 20 to receive HIV-DNA or placebo intradermally (id) 1 mg or 3.8 mg intramuscularly (im) at 0, 1 and 3 months with a needle free device and were boosted with HIV-MVA 10-pfu or placebo im at 9 and 21 months. The vaccine regimen was safe and induced strong and potent immune responses. In this thesis we further explored the immune responses elicited by the HIV-DNA and HIV-MVA vaccine regimen and the effect of boosting with a third HIV-MVA or an envelope protein. In study I, we evaluated the HIV vaccine-induced antibody responses in sera collected from 29 HIVIS03 vaccinees at baseline and four weeks after the second HIV-MVA. High titers of neutralizing antibodies (NAbs) (median 357) were detected using an infectious molecular clone (IMC)/peripheral blood mononuclear cell (PBMC) assay. The NAbs were significantly (but not completely) removed upon depletion of natural killer (NK) cells from PBMC (p=0.0039), indicating a role for Fc–receptor mediated antibody function. ADCC-mediating antibodies were demonstrated in the majority (97%) of the vaccinees against CRF01_AE and/or subtype B. The magnitude of ADCC-mediating antibodies against CM235 CRF01_AE IMC-infected cells correlated with NAbs against CM235 in the IMC/PBMC assay. In studies II and III we explored the duration of immune responses in individuals primed with HIV-DNA and boosted with HIV-MVA in the HIVIS03 trial and the effect of a late third HIV-MVA. Twenty volunteers who had previously received three HIV-DNA and two HIV-MVA immunizations in the HIVIS03 trial were given a third HIV-MVA, three years after the second HIV-MVA boost (HIVIS06). A high proportion of vaccinees showed durable binding antibodies, 90% to HIV-1 subtype C gp140 (median titer 200) and 85% to subtype B gp160 (median titer 100) three years after the second HIV-MVA. The majority of vaccinees had detectable ADCC–mediating antibodies, 70% against CRF01_AE virus–infected cells (median titer 239) and 84% against CRF01_AE gp120–coated cells (median titer 499). Furthermore, 74% of vaccinees still had IFN-γ ELISpot responses, 63% to Gag and 42% to Env. After the third HIV-MVA, there was an increase in Env-binding antibodies and ADCC-mediating antibodies relative to the response seen at the 3-years time point. The frequency of IFN-γ ELISpot responses increased to 95% against Gag or Env, and 90% to both Gag and Env, p = 0.064 and p = 0.002, respectively, after the third HIV-MVA. All 19 (100%) evaluable vaccinees had IgG antibodies to V1V2 CRF01_AE A244 after the second HIV-MVA with a median titer of 3200. A high proportion (75%) of the vaccinees still had V1V2 IgG antibodies to CRF01_AE A244 three years after the second HIV-MVA, which increased to 95% after the third HIV-MVA. The magnitude of response before and after the third MVA increased significantly from a median titer of 400 to 1600, p<0.0001, but not to the same level as after the second HIV-MVA (p=0.025). Surface plasmon resonance/Biacore analysis data supported the ELISA findings. V1V2specific IgG1 antibody responses were more frequently detected than V1V2-specific IgG3 antibodies. AntiV1V2 IgG1 responses decreased after three years but could be boosted by the third HIV-MVA in the majority of the vaccinees. In study IV, we evaluated the safety and impact of boosting with subtype C CN54rgp140 Env protein adjuvanted in glucopyranosyl lipid A-aqueous formulation (GLA-AF) in volunteers previously given three HIV-DNA, followed by two HIV-MVA in the TaMoVac 01 trial. Forty volunteers (35 vaccinees and five placebo recipients) were given two CN54rgp140/ GLA-AF immunizations 30–71 weeks after the second HIV-MVA. The vaccine was safe and well tolerated, except for one incident of asymptomatic hypoglycemia. After the second HIV-MVA vaccination, 34 (97%) of the vaccinees developed Env-specific binding antibodies, whereas 79% and 84% exhibited IFN-γ ELISpot responses to Gag and Env, respectively. Binding antibodies to subtype C, B and CRF01_AE Env were significantly boosted by the CN54rgp140/GLA-AF immunizations, while functional antibodies were not significantly boosted. In contrast, T-cell proliferative responses to subtype B MN antigen and IFN-γ ELISpot responses to Env peptides were significantly enhanced. In conclusion, the HIV-DNA prime/HIV-MVA boost regimen elicited potent antibody and cellular immune responses with remarkable durability, and a third HIV-MVA significantly boosted both antibody and cellular immune responses. Binding antibody responses and Env-specific cell-mediated immune responses but not functional antibody responses, increased after boosting with two CN54rgp140/GLA-AF immunizations following priming with HIV-DNA and HIV-MVA. LIST OF SCIENTIFIC PAPERS I. Joachim A, Nilsson C, Aboud S, Bakari M, Lyamuya EF, Robb ML, Marovich MA, Earl P, Moss B, Ochsenbauer C, Wahren B, Mhalu F, Sandström E, Biberfeld G, Ferrari G, Polonis VR. Potent functional antibody responses elicited by HIV-1 DNA priming and boosting with heterologous HIV-1 recombinant MVA in healthy Tanzanian adults. PLoSONE 2015 10 (4): e0118486. II. Joachim A*, Munseri P*, Nilsson C, Bakari M, Aboud S, Lyamuya EF, Tecleab T, Liankia V, Scarlatti G, Robb ML, Earl P, Moss B, Wahren B, Mhalu F, Ferrari G, Sandström E, Biberfeld G. Three-year durability of immune responses induced by HIV-DNA and HIV-modified vaccinia virus Ankara and effect of a late HIV-MVA in Tanzanian volunteers. AIDS Res Hum Retroviruses 2017. Epub ahead of print doi: 10.1089/AID.2016. 0251 III. Joachim A, Nilsson C, Msafiri F, Onkar S, Munseri P, Aboud S, Lyamuya EF, Bakari M, Billings E, Robb ML, Wahren B, Mhalu F, Sandström E, Rao M, Biberfeld G. Frequent and durable V1V2 antibody responses induced by HIV-1 DNA priming followed by HIV-MVA boosting in healthy Tanzanian volunteers. Manuscript IV. Joachim A*, Bauer A*, Joseph S, Geldmacher C, Munseri P, Aboud S, Missanga M, Mann P, Wahren B, Ferrari G, Polonis V, Robb ML, Weber J, Tatoud R, Maboko L, Hoelscher M, Lyamuya EF, Biberfeld G, Sandström E, Kroidl A, Bakari M, Nilsson C, McCormack S. Boosting with subtype C CN54rgp140 protein adjuvanted with glucopyranosyl lipid adjuvant after priming with HIV-DNA and HIV-MVA is safe and enhances immune responses: a phase I trial. PLoSONE 2016 11(5): e0155702 CONTENTS 1 GENERAL BACKROUND . 1 1.