Expression and Cellular Immunogenicity of a Transgenic Antigen Driven by Endogenous Poxviral Early Promoters at Their Authentic Loci in MVA

CD8+ T cell responses to vaccinia virus are directed almost exclusively against early gene products. The attenuated strain modified vaccinia virus Ankara (MVA) is under evaluation in clinical trials of new vaccines designed to elicit cellular immune responses against pathogens including Plasmodium spp., M. tuberculosis and HIV-1. All of these recombinant MVAs (rMVA) utilize the well-established method of linking the gene of interest to a cloned poxviral promoter prior to insertion into the viral genome at a suitable locus by homologous recombination in infected cells. Using BAC recombineering, we show that potent early promoters that drive expression of non-functional or non-essential MVA open reading frames (ORFs) can be harnessed for immunogenic expression of recombinant antigen. Precise replacement of the MVA orthologs of C11R, F11L, A44L and B8R with a model antigen positioned to use the same translation initiation codon allowed early transgene expression similar to or slightly greater than that achieved by the commonly-used p7.5 or short synthetic promoters. The frequency of antigen-specific CD8+ T cells induced in mice by single shot or adenovirus-prime, rMVA-boost vaccination were similarly equal or marginally enhanced using endogenous promoters at their authentic genomic loci compared to the traditional constructs. The enhancement in immunogenicity observed using the C11R or F11L promoters compared with p7.5 was similar to that obtained with the mH5 promoter compared with p7.5. Furthermore, the growth rates of the viruses were unimpaired and the insertions were genetically stable. Insertion of a transgenic ORF in place of a viral ORF by BAC recombineering can thus provide not only a potent promoter, but also, concomitantly, a suitable insertion site, potentially facilitating development of MVA vaccines expressing multiple recombinant antigens

Generation of multivalent MVA vaccines for malaria

Modified vaccinia virus Ankara (MVA) has been used extensively as a recombinant vector for delivery of antigens from diverse pathogens. Its ability to generate strong antigen specific CD8+ T cell responses in humans has been shown in clinical trials of novel vaccines against malaria, tuberculosis, HIV I AIDS, influenza and cancer. The work in this thesis describes the use of BAC recombineering technology to harness the endogenous regulatory signal (promoter) that drives the expression of non-essential open reading frames (ORFs) in MVA for immunogenic expression of a recombinant antigen. Replacement of the ORFs of four non-essential genes in MVA; C11R, F11l, A44L and B8R with an epitope tagged luciferase positioned to use the same endogenous promoter showed early transgene expression equal to or slightly higher than traditional p7.5 and short synthetic promoter (SSP) constructs. The frequency of antigen-specific CD8+ T cell induced in mice by single dose MVA or adenovirus-prime, rMVA-boost vaccination showed equivalent or slightly higher responses by the endogenous promoters compared to the traditional p7.5 and SSP constructs. Assessment of the growth rate of these viruses showed they were unimpaired and the insertions were genetically stable. Furthermore, the endogenous promoter driven insertion loci of B8R and C11R were used for the construction of a bivalent MVA expressing an epitope tagged luciferase (rLucPb9) and a Photinus pyralis (pLuc) luciferase. The frequency of antigen-specific CD8+ T cells induced in mice by bivalent MV A was equivalent to single-pLuc and single-Pb9 recombinants co-administered as a mixture, at separate sites or administered alone following single dose MV A vaccination but slightly lower for Pb9-specific CD8+ T cell following adenovirus-prime, rMVA-boost.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Generation of multivalent recombinant MVA vaccines for malaria

Modified vaccinia virus Ankara (MVA) has been used extensively as a recombinant vector for delivery of antigens from diverse pathogens. Its ability to generate strong antigen specific CD8+ T cell responses in humans has been shown in clinical trials of novel vaccines against malaria, tuberculosis, HIV/AIDS, influenza and cancer. The work in this thesis describes the use of BAC recombineering technology to harness the endogenous regulatory signal (promoter) that drives the expression of non-essential open reading frames (ORFs) in MVA for immunogenic expression of a recombinant antigen. Replacement of the ORFs of four non-essential genes in MVA; C11R, F11L, A44L and B8R with an epitope tagged luciferase positioned to use the same endogenous promoter showed early transgene expression equal to or slightly higher than traditional p7.5 and short synthetic promoter (SSP) constructs. The frequency of antigen-specific CD8+ T cell induced in mice by single dose MVA or adenovirus-prime, rMVA-boost vaccination showed equivalent or slightly higher responses by the endogenous promoters compared to the traditional p7.5 and SSP constructs. Assessment of the growth rate of these viruses showed they were unimpaired and the insertions were genetically stable. Furthermore, the endogenous promoter driven insertion loci of B8R and C11R were used for the construction of a bivalent MVA expressing an epitope tagged luciferase (rLucPb9) and a Photinus pyralis (pLuc) luciferase. The frequency of antigen-specific CD8+ T cells induced in mice by bivalent MVA was equivalent to single-pLuc and single-Pb9 recombinants co-administered as a mixture, at separate sites or administered alone following single dose MVA vaccination but slightly lower for Pb9-specific CD8+ T cell following adenovirus-prime, rMVA-boost

Potential use of microarray patches for vaccine delivery in low- and middle- income countries

Microarray patches (MAPs), also referred to as microneedle patches, are a novel methodology that have the potential to overcome barriers to vaccine delivery in low- and middle-income countries (LMICs), and transform the way that vaccines are delivered within immunization programs. The World Health Organization’s Initiative for Vaccine Research and its partners are working to understand how MAPs could ease vaccine delivery and increase equitable access to vaccines in LMICs. Global stakeholders have been engaged to evaluate technical, economic, and programmatic challenges; to validate assumptions where possible; and to propose areas of focus to facilitate future vaccine-MAP product development. This report summarizes those learnings

Schematic of transgene insertion at endogenous promoter driven locus of MVA, using <i>B8R</i> as an example.

<p>(A) Surrounding open reading frames (ORFs) in the MVA genome are indicated by white arrows, with <i>B8R</i> highlighted in grey. Black “p” above black bar indicates predicted <i>B8R</i> early promoter core region (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040167#pone-0040167-t002" target="_blank">Table 2</a>), overlapping with the <i>B7R</i> ORF. The left homology arm (LHA) and right homology arm (RHA) sequences (white boxes), each 50 bp in length, were added by PCR to the ends of a cassette comprising a model antigen, tPA-Pb9-rLuc8PV (narrow grey arrow) and the bacterial selectable marker <i>GalK</i> (black arrow), with its bacterial promoter (small black triangle). The LHA was designed to place the initiation codon of tPA-Pb9-rLuc8PV in the same position as that of the <i>B8R</i> ORF. Crossed lines indicate homology arm recombination events between targeting amplicon and MVA-BAC. (B) After recombineering of this 2.4 kb targeting amplicon into MVA-BAC to replace <i>B8R</i>, tPA-Pb9-rLuc8PV was placed under control of the <i>B8R</i> promoter.</p

Timecourse of endogenous promoter activities compared to p7.5 and SSP <i>in vitro</i>.

<p>BHK cells were “spinoculated” (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040167#s2" target="_blank">Materials and Methods</a>) with 1 pfu/cell of recombinant MVA carrying tPA-Pb9-rLuc8PV under the control of the indicated promoters. Renilla luciferase activity was quantified in the inoculum, the culture supernatant at various time points post-infection, and in the cell lysate at 24 h post-infection. Cells were either untreated (A) or exposed to 40 µM AraC during and after infection (B), to inhibit post-replicative gene expression. Data shown are the mean and standard deviation of duplicates and are representative of two independent experiments.</p

Genetic stability of rMVA expressing tPA-Pb9-rLuc8PV under control of endogenous promoters.

<p>Viruses were subjected to ten serial passages in CEFs, titred, and inoculated onto BHK cells at 0.001 pfu/cell. After 2 days, the cells were harvested and individually sorted into the wells of a 96-well plate using the CyCLONE attachment of a MoFlo flow cytometer. Two days later, renilla luciferase activity in the cell lysates was determined after scoring of wells as positive (+) or negative (−) for the viral GFP marker gene, indicating infection in the well. A cut-off of three standard deviations above the geometric mean of the GFP (dashed line labelled 3σ) was used to score GFP⁺ and GFP⁻ wells luciferase positive (Luc⁺) or negative (Luc⁻). Wells in which cell monolayers were lost during processing were excluded. Raw data for the pB8R recombinant (A) and well scores for all viruses (B) are shown.</p

Sequences of promoters, with transcriptional and translational (ATG) start sites shown capitalised, and the predicted early promoter core sequences underlined [19].

a<p>The italicised ‘t’ in <i>F11L</i> was mutated during recombineering to identity with vaccinia virus Western Reserve strain (VACV-WR).</p>b<p>The second (downstream) ATG was used in the recombinant viruses described in the text. This is the ATG of the ORF as originally annotated in MVA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040167#pone.0040167-Antoine1" target="_blank">[43]</a>. The upstream ATG, however, likely represents the authentic translational start site in vaccinia virus, encoding a protein that is severely truncated in MVA by a small deletion (see text).</p>c<p>Conventional insertion of promoter linked to ORF at the TK locus of MVA. Dashes indicate appendage of multiple cloning site (MCS).</p

Figure 5

<p>(A) Timecourse of activity of mH5 promoter compared to p7.5 <i>in vitro</i>. BHK cells were “spinoculated” (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040167#s4" target="_blank">Materials and Methods</a>) with 1 pfu/cell of recombinant MVA carrying tPA-Pb9-rLuc8PV under the control of the indicated promoters. Renilla luciferase activity was quantified in the inoculum and then in the culture supernatant at various time points post-infection. Cells were either untreated or exposed to 40 µM AraC during and after infection as indicated, to inhibit post-replicative gene expression. Data shown are the mean and standard deviation of duplicates. The data shown are representative of two independent experiments. (B) Cellular immunogenicity of recombinant MVA antigen (tPA-Pb9-rLuc8PV) driven by mH5 compared to p7.5 in a single-shot vaccination regimens. BALB/c mice were immunized i.m. with 10⁶ pfu rMVA and splenic CD8⁺ T cell responses to Pb9 peptide were determined 7 days later by intracellular cytokine staining and flow cytometry. Circles represent the responses of individual mice, with lines indicating the mean and the error bars showing SEM. See text for statistical analysis. The data shown are representative of two independent experiments.</p

Loci selected for insertion of endogenous promoter driven transgene by replacement of MVA open reading frame (ORF).

*<p>Designations in brackets refer to categorisation into immediate early (IE) or early (E) clusters by microarray analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040167#pone.0040167-Assarsson1" target="_blank">[20]</a>, and into the corresponding E1.1 and E1.2 clusters by deep sequencing <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040167#pone.0040167-Yang1" target="_blank">[19]</a>, of vaccinia virus mRNA.</p