Investigation of the prevalence and role of mobile genetic elements associated with an aminoglycoside resistance gene, aacC2a, in Acinetobacter baumannii

Includes abstract. Includes bibliographical references (leaves 115-141)

Construction and evaluation of three candidate vaccines expressing HIV-1 subtype-C mosaic Gag

Includes bibliographical referencesOf the 35 million people living with HIV-1 globally, approximately 71.4% are in the resource-limited sub-Saharan Africa. The immense sequence diversity of HIV-1, even within subtypes, makes it challenging to develop effective vaccines that target a wide range of HIV subtypes. Mosaic immunogens have been computationally designed to specifically overcome this hurdle by maximizing the inclusion of common T cell epitopes. When compared to consensus immunogens, polyvalent mosaic immunogens of HIV-1 group M have shown increased breadth and depth of antigen-specific T-cell responses. More than 90% of HIV positive individuals in sub-Saharan Africa are infected with HIV-1 subtype C (HIV-1C). We therefore designed, constructed, and evaluated candidate vaccines expressing HIV-1C mosaic Gag (GagM) in a proof of concept study. Gag was chosen as the most appropriate target for a T cell-based vaccine as there are many studies correlating control of HIV viral load with T cell responses to Gag. The immunogen was designed by Fischer et al., 2007 (1). Three different vaccine platforms were chosen based on their different strengths to be used in prime-boost regimens to determine the immunogenicity of HIV-1C GagM in mice. The first was a pantothenic auxotroph of the tuberculosis vaccine Mycobacterium bovis Bacille Calmette Guérin (BCG). The second was a DNA vaccine vector with enhanced expression of transgenes due to a novel enhancer element from porcine circovirus type 1, which has been demonstrated to increase gene expression. The third vaccine vector selected was the well characterised poxvirus modified vaccinia Ankara (MVA)

HIV-1 subtype C mosaic Gag expressed by BCG and MVA elicits persistent effector t cell responses in a prime-boost regimen in mice

Over 90% of HIV/AIDS positive individuals in sub-Saharan Africa are infected with highly heterogeneous HIV-1 subtype C (HIV-1C) viruses. One of the best ways to reduce the burden of this disease is the development of an affordable and effective prophylactic vaccine. Mosaic immunogens are computationally designed to overcome the hurdle of HIV diversity by maximizing the expression of potential T cell epitopes. Mycobacterium bovis BCG Δ panCD auxotroph and modified vaccinia Ankara (MVA) vaccines expressing HIV-1C mosaic Gag (Gag M ) were tested in a prime-boost regimen to demonstrate immunogenicity in a mouse study. The BCG-Gag M vaccine was stable and persisted 11.5 weeks post vaccination in BALB/c mice. Priming with BCG-Gag M and boosting with MVA-Gag M elicited higher Gag-specific IFN-γ ELISPOT responses than the BCG-Gag M only and MVA-Gag M only homologous vaccination regimens. The heterologous vaccination also generated a more balanced and persistent CD4 + and CD8 + T cell Gag-specific IFN-γ ELISPOT response with a predominant effector memory phenotype. A Th1 bias was induced by the vaccines as determined by the predominant secretion of IFN-γ, TNF-α, and IL-2. This study shows that a low dose of MVA (10 4 pfu) can effectively boost a BCG prime expressing the same mosaic immunogen, generating strong, cellular immune responses against Gag in mice. Our data warrants further evaluation in non-human primates. A low dose vaccine would be an advantage in the resource limited countries of sub-Saharan Africa and India (where the predominating virus is HIV-1 subtype C)

Immunological evaluation of a DNA-Gag^M prime/MVA-Gag^M boost in BALB/c mice.

<p>(<b>A</b>) Vaccination schedule used. (<b>B</b>) Cumulative IFN-γ ELISPOT CD8⁺ and CD4⁺ responses of vaccinated mice to HIV-1 Gag peptides. The ELISPOT assay was done using pooled spleens on the day of sacrifice using three Gag-specific peptides for stimulation. Bars are the mean and standard deviation of the mean responses for the indicated individual peptides from 3 independent experiments. Responses are expressed as sfu/10⁶ splenocytes after background subtraction. Horizontal bars with asterisks indicate statistical significance of the mean responses between the indicated groups. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001; Student t-test of unpaired data. Total frequency of CD8+ (<b>C</b>) and CD4+ (<b>D</b>) T cells producing IFN-γ, IL-2, and/or TNF-α in response to HIV-1 Gag peptide stimulation. ICS and flow cytometry were carried out on pooled spleens per group using three Gag-specific peptides for stimulation. The memory distribution of the cytokine producing T-cells in the central and effector memory compartment (T_CM and T_EM) are represented as pie charts above each corresponding bar per group. Cells were positive for cytokine production if the proportion was ≥ 0.05% after subtracting the background. The cellular phenotype was positive if there were ≥ 10 cells per test.</p

Heterologous prime-boost vaccination with DNA and MVA vaccines, expressing HIV-1 subtype C mosaic Gag virus-like particles, is highly immunogenic in mice - Fig 1

<p><b>p24 Gag production by:</b> (<b>A</b>) <b>DNA vaccines in HEK cells.</b> HEK cells were transfected with 4μg of DNA-Gag^M or DNA-Gag^N and samples taken at the indicated time points. (<b>B</b>) <b>Recombinant MVA in BHK-21 and HeLa cell lines.</b> Permissive (BHK-21) and non-permissive (HeLa) cell lines were infected at an MOI of 0.1 with MVA-Gag^M and samples taken at the indicated time points. Gag p24 was detected using an ELISA assay.</p

Amino acid sequence alignment of HIV-1 subtype C mosaic Gag and strain Du422 Gag.

<p>The P17 (matrix), P24 (capsid), SP-1(spacer region 1), P7 (nucleocapsid), SP-2 (spacer region 2) and P6 domains of Gag are shown. The PTAPP binding site for ESCRT-1 protein, TSG101, is indicated as is the LxxLF motif which interacts with ALIX protein of ESCRT-III. Mouse Gag CD8+ (AMQMLKDTI) and CD4+ (NPPIPVGRIYKRWIILGLNK GagCD4(13) and FRDYVDRFFKTLRAEQATQE GagCD4(17)) epitopes are indicated in black, bold font.</p

Mouse immunization regimen.

<p>Mouse immunization regimen.</p

Recombinant MVA construction.

<p>The transfer vector pTJMVA2 was designed to insert the <i>gag</i>^<i>M</i> gene under the transcriptional control of the mH5 promoter, between the MVA A11R and A12L ORFs. The selection (<i>bsd</i>) and marker (<i>gfp</i>) genes were expressed as a fused protein under the transcriptional control of the pSS MVA synthetic promoter.</p

Schematic representation of the BCG shuttle vectors and the determination of their genetic integrity before and after vaccination.

<p>Schematic representation of (<b>A</b>) pTJBCG3, which was used to make the BCG-Gag^M vaccine, (<b>B</b>) pEM19 which was used to make the BCG^E vaccine. Restriction sites used for cloning and restriction mapping analysis are indicated in black bold type. OriE–<i>E</i>. <i>coli</i> origin of replication; OriM–mycobacterial origin of replication, 19kD ss– 19kD signal sequence; KanR–kanamycin resistance gene. (<b>C</b>) pTJBCG3 digested with <i>Xho</i>I. Lane 1 is a positive control of pTJBCG3 DNA prior to transformation into BCGΔ<i>panCD</i>. Lanes 2–21 contain pTJBCG3 DNA obtained from recombinant BCG-Gag^M vaccine stocks. (<b>D</b>) pEM19 digested with <i>Sma</i>I. Lanes 1–15 contain pEM19 plasmid DNA isolated from recombinant BCG^E vaccine stocks, and lane 16 pEM19 plasmid DNA isolated prior to transformation into BCGΔ<i>panCD</i> (positive control). PCR amplification of DNA from rBCG obtained from the spleens and lymph nodes of mice vaccinated with BCG-Gag^M (Group 2) (<b>E</b>) or BCG^E (Group 5) (<b>F</b>) 11.5 weeks post vaccination. Lane 1 –negative control; Lane 2 –positive control; Lane 3–12 are PCR products from rBCG isolated from homogenised spleen (3–7) or lymph nodes (8–12). Lanes M in <b>C</b>—<b>F</b> contain the molecular weight marker O’GeneRuler^TM 1kb DNA ladder.</p

<i>In vitro</i> expression of Gag by BHK-21 cells infected with MVA-Gag^M.

<p>(<b>A</b>) BHK-21 cells were infected with MVA-Gag^M, wild type MVA (wtMVA), or left uninfected for 72 hours. HIV-1 Gag was detected with anti-Gag antibody ARP432 (α-Gag; top panels) as well as an anti-VACV antibody (α-MVA; bottom panels), followed by an anti-rabbit HRP-conjugated antibody. (<b>B</b>) Cell lysates were prepared from BHK-21 cells infected with MVA-Gag^M (lane 4), wild type MVA (lane 3), or left uninfected (lane 2). BHK cells transfected with a plasmid known to express full length Gag was used as a positive control (lane 1). Western blots were probed with a rabbit anti-HIV-1-p24 Gag antibody (ARP432), followed by an anti-rabbit antibody conjugated to alkaline phosphatase (Sigma-Aldrich, USA). A Precision Plus Protein Kaleidoscope pre-stained standard (lane M; Biorad, USA) was used and the sizes are indicated on the left.</p