Evaluation of HIV testing strategies and monitoring of immune responses in HIV-vaccinated individuals in Tanzania

This thesis describes studies on the evaluation of human immunodeficiency virus (HIV) enzyme-linked immunosorbent assays (ELISAs) and simple rapid HIV assays for use in HIV testing strategies in resource-limited settings and studies of HIV vaccine-induced immune responses. Peripheral blood mononuclear cell (PBMC) preparation techniques were also studied in preparation for use in the HIV vaccine trials.

The performance of two antibody ELISAs (Vironostika Uni-Form II plus O and Enzygnost anti-HIV-1/2 Plus) and two new diagnostic HIV antigen/antibody combination ELISAs (Murex and Vironostika HIV Uni-Form II antigen/antibody) was evaluated using 1380 serum samples from Tanzanian individuals (paper I). The sensitivity at initial testing was 100% for all assays except Vironostika Uni-Form II plus O which showed one false negative sample at initial testing but 100% sensitivity after repeat testing. The initial specificity was 99.8% for Enzygnost, 98.9% for each of the antigen/antibody ELISAs and 97.0% for Vironostika Plus O ELISA. An alternative confirmatory HIV testing strategy based on initial testing on any of the two antigen/antibody assays followed by testing of reactive samples on the Enzygnost anti-HIV-1/2 Plus assay gave 100% specificity (95% CI; 99.7-100%).

The performance of five simple rapid HIV antibody assays was evaluated using 1433 whole blood samples (paper II). The sensitivity at initial testing of Determine, SD Bioline and Uni-Gold was 100% while First Response and Stat-Pak had a sensitivity of 99.5% and 97.7%, respectively, which increased to 100% on repeat testing. The initial specificity of the Uni-Gold assay was 100% while the specificities were 99.6%, 99.4%, 99.6% and 99.8% for Determine, SD Bioline, First Response and Stat-Pak assays, respectively. An alternative confirmatory HIV testing strategy based on initial testing on SD Bioline followed by testing of reactive samples on the Determine gave 100% sensitivity (95% CI; 99.1-100) and 100% specificity (95% CI; 96-99.1) with Uni-Gold as tiebreaker for discordant results and was adopted as a national algorithm in Tanzania.

Standard Ficoll-Paque gradient (FIP) centrifugation, BD vacutainer cell preparation tube (CPT) and Greiner Bio-One LeucoSep tube techniques for PBMC preparation were evaluated (paper III). No differences in mean recovery or mean viability of fresh PBMCs were observed between FIP centrifugation and CPT techniques used in Stockholm. In Dar es Salaam, recovery and viability of PBMCs isolated by FIP technique was higher compared to CPT purified cells. LeucoSep cell separation gave a higher yield and viability than FIP cell separation. The cells purified by the different techniques at the two sites performed equally well in interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISpot) assays.

In a phase 1 HIV-1 DNA prime MVA boost vaccine trial in Sweden (HIVIS01/02), HIV-specific lymphoproliferative responses were tested by a [3H]-thymidine uptake assay and a flow-cytometric assay using whole blood (FASCIA-WB) (paper IV). A FASCIA using PBMC (FASCIA-PBMC) was also employed (n=14).Two weeks after the HIV-MVA boost 35 of 38 (92%) vaccinees were reactive by the thymidine uptake assay. Thirty-two of 38 (84%) vaccinees were reactive by the CD4+ T-cell FASCIA-WB, and 7 of 38 (18%) also exhibited CD8+ T-cell responses. There was strong correlation between the proliferative responses measured by the thymidine uptake assay and CD4+ T-cell FASCIA-WB (r=0.68; P < 0.01). Fourteen vaccinees were analyzed using all three assays. Ten of 14 (71%) and 11/14 (79%) demonstrated CD4+ T-cell responses in FASCIA-WB and FASCIA-PBMC, respectively. CD8+ T-cell reactivity was observed in 3/14 (21%) and 7/14 (50%) using the FASCIA-WB and FASCIA-PBMC, respectively. All 14 were reactive by the thymidine uptake assay. A FASCIA-PBMC, which allows simultaneous phenotyping, may be an option to the [3H] thymidine uptake assay for assessment of vaccine-induced T-cell proliferation, especially in isotope-restricted settings.

In the HIVIS03 phase I/II HIV vaccine trial in Tanzania, sixty HIV-uninfected volunteers randomised to three groups of 20, received DNA plasmid vaccine 1 mg intradermally (id) or 3.8 mg intramuscularly (im) or placebo using a needle-free injection device (paper V). DNA plasmids vectoring HIV-1 genes gp160 subtypes A, B, C; rev B; p17/p24 gag A, B and Rtmut B were given at weeks 0, 4 and 12. Recombinant MVA (10[8] pfu) expressing HIV-1 Env, Gag, Pol of CRF01_AE or placebo was administered im at month 9 and 21. The vaccines were well tolerated. Two weeks after the first HIV-MVA boost 35/35 (100%) vaccinees had IFN-γ ELISpot responses; 35 (100%) to Gag and 31 (89%) to Env. Two to four weeks after the second HIV-MVA boost, 28/29 (97%) vaccinees had IFN-γ responses. The id-primed recipients had significantly higher responses to Env than im recipients after HIV-MVA boost. Intracellular cytokine staining for Gag-specific IFN-γ/IL-2 production showed both CD8+ and CD4+ T-cell responses. All vaccinees had HIV-specific lymphoproliferative responses. All vaccinees reacted in diagnostic HIV serological tests and 26/29 (90%) had antibodies against gp160 after the second HIV-MVA boost. A high neutralizing antibody response rate (31-83% depending on the clade B or AE virus tested) was demonstrated using a PBMC assay. In conclusion, this vaccine approach was safe and highly immunogenic.

List of scientific papers

I. Aboud S, Urassa W, Lyamuya E, Mhalu F, Biberfeld G. Evaluation of HIV antibody and antigen/antibody combination ELISAs for use in an alternative confirmatory HIV testing strategy in Dar es Salaam, Tanzania. J Virol Methods. 2006 August; 135(2):192–196.
https://doi.org/10.1016/j.jviromet.2006.03.005

II. Lyamuya EF, Aboud S, Urassa WK, Sufi J, Mbwana J, Ndugulile F, Massambu C. Evaluation of simple rapid HIV assays and development of national rapid HIV test algorithms in Dar es Salaam, Tanzania. BMC Infect Dis. 2009 February; 9(1):19.
https://doi.org/10.1186/1471-2334-9-19

III. Nilsson C, Aboud S, Karlen K, Hejdeman B, Urassa W, Biberfeld G. Optimal blood mononuclear cell isolation procedures for gamma interferon enzyme-linked immunospot testing of healthy Swedish and Tanzanian subjects. Clin Vaccine Immunol. 2008 April; 15(4): 585-589.
https://doi.org/10.1128/CVI.00161-07

IV. Aboud S, Nilsson C, Karlen K, Marovich M, Wahren B, Sandstrom E, Gaines H, Biberfeld G, Godoy-Ramirez K. Strong HIV-specific CD4+ and CD8+ T lymphocyte proliferative responses in healthy individuals immunized with an HIV-1 DNA vaccine and boosted with HIV-1 recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 genes. Clin Vaccine Immunol. 2010 July; 17(7):1124-1131.
https://doi.org/10.1128/CVI.00008-10

V. Bakari M, Aboud S, Nilsson C, Francis J, Buma D, Moshiro C, Aris EA, Lyamuya EF, Janabi M, Earl P, Robb M, Marovich M, Wahren B, Pallangyo K, Biberfeld G, Mhalu F, Sandström E, for the HIVIS study group. Broad and potent immune responses to a low dose of intradermal HIV-1 DNA boosted with HIV-1 recombinant MVA among healthy adults in Tanzania. Vaccine. [Accepted]
https://doi.org/10.1016/j.vaccine.2011.08.001