Oral vaccination with a recombinant Salmonella vaccine vector provokes systemic HIV-1 subtype C Gag-specific CD4+ Th1 and Th2 cell immune responses in mice

<p>Abstract</p> <p>Background</p> <p>Recombinant <it>Salmonella </it>vaccine vectors may potentially be used to induce specific CD4+ T cell responses against foreign viral antigens. Such immune responses are required features of vaccines against pathogens such as human immunodeficiency virus type 1 (HIV-1). The aim of this study was to investigate the induction of systemic HIV-1-specific CD4+ T helper (Th) responses in mice after oral immunization with a live attenuated <it>Salmonella </it>vaccine vector that expressed HIV-1 subtype C Gag. Groups of BALB/c mice were vaccinated orally three times (4 weeks apart) with this recombinant <it>Salmonella</it>. At sacrifice, 28 days after the last immunization, systemic CD4+ Th1 and Th2 cytokine responses were evaluated by enzyme-linked immunospot assay and cytometric bead array. HIV-1 Gag-specific IgG1 and IgG2a humoral responses in the serum were determined by enzyme-linked immunosorbent assay.</p> <p>Results</p> <p>Mice vaccinated with the recombinant <it>Salmonella </it>elicited both HIV-1-specific Th1 (interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α)) and Th2 (interleukin-4 (IL-4) and interleukin-5 (IL-5)) cytokine responses. The vaccine induced 70 (IFN-γ) spot-forming units (SFUs)/10e6 splenocytes and 238 IL-4 SFUs/10e6 splenocytes. Splenocytes from vaccinated mice also produced high levels of Th1 and Th2 cytokines upon stimulation with a Gag CD4 peptide. The levels of IFN-γ, TNF-α, IL-4 and IL-5 were 7.5-, 29.1-, 26.2- and 89.3-fold above the background, respectively. Both HIV-1 Gag-specific IgG1 and IgG2a antibodies were detected in the sera of vaccinated mice.</p> <p>Conclusion</p> <p>The study highlights the potential of orally-delivered attenuated <it>Salmonella </it>as mucosal vaccine vectors for HIV-1 Subtype C Gag to induce Gag-specific CD4+ Th1 and Th2 cellular immune responses and antibodies which may be important characteristics required for protection against HIV-1 infection.</p

An oral recombinant Salmonella enterica serovar Typhimurium mutant elicits systemic antigen-specific CD8+ T cell cytokine responses in mice

<p>Abstract</p> <p>Background</p> <p>The induction of antigen-specific CD8+ T cell cytokine responses against an attenuated, oral recombinant <it>Salmonella enterica </it>serovar Typhimurium vaccine expressing a green fluorescent protein (GFP) model antigen was investigated. A GFP expression plasmid was constructed in which the <it>gfp </it>gene was fused in-frame with the 5' domain of the <it>Escherichia coli β</it>-galactosidase <it>α</it>-gene fragment with expression under the <it>lac </it>promoter. Groups of mice were orally immunized three times with the bacteria and systemic CD8+ T cell cytokine responses were evaluated.</p> <p>Results</p> <p>High level of the GFP model antigen was expressed by the recombinant <it>Salmonella </it>vaccine vector. Systemic GFP-specific CD8+ T cell cytokine (IFN-γ and IL-4) immune responses were detected after mice were orally vaccinated with the bacteria. It was shown that 226 net IFN-γ and 132 net IL-4 GFP-specific SFUs/10e6 splenocytes were formed in an ELISPOT assay. The level of IFN-γ produced by GFP peptide-stimulated cells was 65.2-fold above background (p < 0.05). The level of IL-4 produced by the cells was 10.4-fold above background (p < 0.05).</p> <p>Conclusion</p> <p>These results suggested that a high expressing recombinant <it>Salmonella </it>vaccine given orally to mice would elicit antigen-specific CD8+ T cell responses in the spleen. <it>Salmonella </it>bacteria may, therefore, be used as potential mucosal vaccine vectors.</p

The use of directed evolution to create a stable and immunogenic recombinant BCG expressing a modified HIV-1 Gag antigen

Numerous features make Mycobacterium bovis BCG an attractive vaccine vector for HIV. It has a good safety profile, it elicits long-lasting cellular immune responses and in addition manufacturing costs are affordable. Despite these advantages it is often difficult to express viral antigens in BCG, which results in genetic instability and low immunogenicity. The aim of this study was to generate stable recombinant BCG (rBCG) that express high levels of HIV antigens, by modification of the HIV genes. A directed evolution process was applied to recombinant mycobacteria that expressed HIV-1 Gag fused to the green fluorescent protein (GFP). Higher growth rates and increased GFP expression were selected for. Through this process a modified Gag antigen was selected. Recombinant BCG that expressed the modified Gag (BCG[pWB106] and BCG[pWB206]) were more stable, produced higher levels of antigen and grew faster than those that expressed the unmodified Gag (BCG[pWB105]). The recombinant BCG that expressed the modified HIV-1 Gag induced 2 to 3 fold higher levels of Gag-specific CD4 T cells than those expressing the unmodified Gag (BCG[pWB105]). Mice primed with 10 7 CFU BCG[pWB206] and then boosted with MVA-Gag developed Gag-specific CD8 T cells with a frequency of 1343±17 SFU/10 6 splenocytes, 16 fold greater than the response induced with MVA-Gag alone. Levels of Gag-specific CD4 T cells were approximately 5 fold higher in mice primed with BCG[pWB206] and boosted with MVA-Gag than in those receiving the MVA-Gag boost alone. In addition mice vaccinated with BCG[pWB206] were protected from a surrogate vaccinia virus challenge

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

Genetic Alteration of Mycobacterium smegmatis To Improve Mycobacterium-Mediated Transfer of Plasmid DNA into Mammalian Cells and DNA Immunization▿

Mycobacteria target and persist within phagocytic monocytes and are strong adjuvants, making them attractive candidate vectors for DNA vaccines. We characterized the ability of mycobacteria to deliver transgenes to mammalian cells and the effects of various bacterial chromosomal mutations on the efficiency of transfer in vivo and in vitro. First, we observed green fluorescent protein expression via microscopy and fluorescence-activated cell sorting analysis after infection of phagocytic and nonphagocytic cell lines by Mycobacterium smegmatis or M. bovis BCG harboring a plasmid encoding the fluorescence gene under the control of a eukaryotic promoter. Next, we compared the efficiencies of gene transfer using M. smegmatis or BCG containing chromosomal insertions or deletions that cause early lysis, hyperconjugation, or an increased plasmid copy number. We observed a significant—albeit only 1.7-fold—increase in the level of plasmid transfer to eukaryotic cells infected with M. smegmatis hyperconjugation mutants. M. smegmatis strains that overexpressed replication proteins (Rep) of pAL5000, a plasmid whose replicon is incorporated in many mycobacterial constructs, generated a 10-fold increase in plasmid copy number and 3.5-fold and 3-fold increases in gene transfer efficiency to HeLa cells and J774 cells, respectively. Although BCG strains overexpressing Rep could not be recovered, BCG harboring a plasmid with a copy-up mutation in oriM resulted in a threefold increase in gene transfer to J774 cells. Moreover, M. smegmatis strains overexpressing Rep enhanced gene transfer in vivo compared with a wild-type control. Immunization of mice with mycobacteria harboring a plasmid (pgp120hE) encoding human immunodeficiency virus gp120 elicited gp120-specific CD8 T-cell responses among splenocytes and peripheral blood mononuclear cells that were up to twofold (P < 0.05) and threefold (P < 0.001) higher, respectively, in strains supporting higher copy numbers. The magnitude of these responses was approximately one-half of that observed after intramuscular immunization with pgp120hE. M. smegmatis and other nonpathogenic mycobacteria are promising candidate vectors for DNA vaccine delivery

rBCG prime and MVA-Gag boost immune responses.

<p>Mice were primed with BCG[pWB206] or BCG[pHS207] (intraperitoneal vaccination; doses of 10⁷ CFU, 10⁵ CFU, 10³ CFU) or left unprimed, then boosted on day 56 with MVA-Gag (intramuscular vaccination, 10⁷ pfu). Splenocytes pooled from a group of 5 mice were used on day 68 in <b>A.</b> an IFN-γ ELISPOT assay with the GFPCD8 peptide, GagCD8 peptide or GagCD4 peptide. Bars represent the average number of SFU/10⁶ splenocytes ± the standard deviation of triplicate reactions after subtraction of average background responses of not more than 20±10 SFU per 10⁶ splenocytes. <b>B.</b> Splenocytes were cultured (48 h) with the GFPCD8 peptide, GagCD8 peptide or GagCD4 peptide and cytokine levels in the culture supernatant were quantified using a Th1/Th2 cytokine bead array assay and flow cytometry. Only IFN-γ was detected in the culture fluid. Levels are expressed as pg/10⁶ splenocytes and are from a representative experiment.</p

CTL responses measured in a ⁵¹Cr release assay.

<p>Splenocytes pooled from a group of 5 mice on day 68 after a prime with BCG[pWB206] or BCG[pHS207] (10⁷ CFU, 10⁵ CFU, 10³ CFU) or no prime and boost with MVA-Gag (10⁷ pfu) on day 56, were stimulated with the GagCD8 peptide for 6 days. Generated effector cells were used in a ⁵¹Cr release assay using p815 antigen presenting cells in the presence and absence of peptide. Data values indicate the mean net percentage Gag-peptide specific lysis ± the standard deviation (n = 3), calculated after the background lysis (<10%) in the absence of peptide has been subtracted. Net Gag-specific lysis was considered positive if >10% and are shown.</p

Maps and sequence of plasmids that express high levels of GFP, generated through mutation and selection.

<p>Position numbers are as for the parental plasmid pWB105.<b>A.</b> Potential ribosome binding site and start site utilized for P24-GFP expression in pWB106 transformants. <b>B.</b> Mutations found in transformants expressing high levels of p24. Mutation type symbols: X, 1 base pair deletion of an A nucleotide at position 4884 that allows expression of p24 from an internal start codon;Y, 1 bp insertion of an A nucleotide between positions 5539–5540; Z, 1 bp deletion of a C nucleotide at position 5706. Deletions (Δ) are shown with positions given to include the remaining portion. <b>C.</b> Map showing plasmids generated by deleting regions of plasmid pWB106 to determine whether p24-GFP is transcribed from the <i>hsp60</i> promoter. GFP fluorescence indicates whether <i>M. smegmatis</i> transformed with these plasmids showed GFP fluorescence.</p