Safety of LSDV in Immunocompromised Mice
All mouse procedures were approved by the University of Cape Town Animal Research Ethics Committee. To test the safety of wild-type LSDV, RAG mice (lacking both T-cells and B-cells) and CD4 T cell knockout mice (6-8 weeks old, 5 per group) were inoculated with 104 and 106 focus forming units (ffu) per mouse by the intramuscular route, with 50 μl injected into each quadricep muscle. Mouse weights and welfare with respect to weight, appetite, coat condition and behaviour were recorded daily for 4 weeks.
DNA vaccine, viruses and cells
The plasmid pVRC-grttnC (manufactured by Aldevron, Fargo, ND, USA) expresses an HIV-1 subtype C polyprotein consisting of Gag, RT, Tat and Nef (grttn) and is a second generation DNA vaccine. It differs from the first generation vaccine pTHgrttnC  in that the pTH vector backbone has been replaced with the pVRC backbone provided by the Vaccine Research Center of the National Institutes of Health, Bethesda, Maryland, USA . The HIV-1 genes (grttn) were modified for safety issues, codon optimized for human expression  and cloned downstream of the cytomegalovirus AD169 immediate-early promoter, with an enhancer intron A and a Kozak sequence.
LSDV Neethling strain, a bovine vaccine, was provided by Onderstepoort Veterinary Institute, South Africa. MVA was obtained from Dr B. Moss (NIH, USA).
Foetal bovine testes cells (FBT) were isolated from trypsinized foetal bovine testes  obtained from the local abattoir. Madin-Darby bovine kidney epithelial (MDBK) cells and Syrian baby hamster kidney (BHK-21) cells were obtained from the American Type Culture Collection (ATCC). Chick embryo fibroblast (CEF) cells were isolated from 10-day old embryonated chicken eggs and were maintained in 2% FCS and DMEM (Gibco™).
Construction of transfer vectors for cloning into LSDV and MVA
Plasmid pLW-51, (kindly provided by Linda Wyatt; NIH, USA), was used as a backbone for the transfer vectors. The HIV-1 genes were derived from recently transmitted HIV-1 subtype C strains in South Africa . For both the LSDV and MVA transfer vectors grttn was cloned directly downstream of the vaccinia virus early/late mH5 promoter. pLW-51grttn:
Grttn was excised from pJH-01  using SalI followed by XmaCI and cloned into the SalI and XmaCI sites of pLW-51 to generate pLW-51grttn. pYS-05: MVA flanking sequences in pLW-51 were replaced with sequences from the non-essential ribonucleotide reductase gene of LSDV . LSDV flank I (LF1) and LSDV flank II (LFII), DNA fragments of 508 bp and 552 bp respectively were PCR amplified from LSDV DNA using the following primer pairs:
LF1 forward: 5'-GAATTCATGGTATAAAATAAAATGGAACC-3';
LF1 reverse: 5'-GCGCGCCAAACGCTATTAATCGTTCTC-3';
LF2 forward: 5'-CTGCAGTTGAGGGAATATTCTTTTCCGG-3' and
LF2 reverse: 5'-AAGCTTGGTTATTCAAGATAATTAACAAGAG-3'.
Restriction enzyme sites EcoRI and BssHII were included at the beginning and end of LFI respectively; PstI and HindIII were added at the beginning and end of LFII respectively. Amplified fragments were cloned into the SmaI site of pUC19 plasmid using a Rapid DNA ligation kit (Roche, Germany). MVA flank I was excised from pLW-51 with EcoRI and BssHII (Roche, Germany) and replaced with LFI excised from pUC19 with the same restriction enzymes. MVA flank II was then replaced with LFII using PstI and HindIII to generate plasmid pYS-03. Grttn  with an upstream Kozak sequence was subcloned from pJH-01  into the SalI and XmaCI sites of pYS-03. The E.coli xanthine-guanine phosphoribosyl transferase gene (Gpt) under the control of the VV p7.5 promoter was excised from pGpt07/14  on an EcoRI fragment, blunt-ended and cloned into the blunt-ended XhoI restriction enzyme site.
Construction of rMVA-grttn
Chick embryo fibroblast (CEF) cells were infected with wt MVA (NIH) (0.1 pfu/cell) and transfected with 500 ng pLW-51-Grttn linearized with NdeI. A lysate was prepared 3 days later and used to infect BHK-21 cells. Nine rounds of plaque picking were performed. The final virus isolate expressed Grttn (indicated by positive immunostaining with an anti-RT antibody(ARP428) from the National Institute for Biological Standards and Control (NIBSC) AIDS reagent program, United Kingdom) and was negative for GUS staining. rMVA-grttn was expanded in eggs (Westwood et al., 1957) and the titre determined in BHK-21 cells. MVA was detected with a rabbit anti-vaccinia antibody (Biogenesis Ltd, United Kingdom) and swine anti-rabbit HRP (Dako, Denmark). Grttn expression was detected with sheep anti-RT (ARP428) and rabbit anti-sheep HRP (Dako, Denmark). Peroxidase was reacted with o-dianisidine (Sigma-Aldrich, USA) in the presence of H2O2 to visualize infected cells. Identical virus titres were obtained irrespective of the antibody used to detect virus-infected cells, indicating all MVA-infected cells expressed Grttn.
Preparation of rLSDV-grttn
FBT cells were infected with LSDV (0.1 ffu per cell) followed by transfection with pYS-05 (400 ng). Twenty-four hours later the medium was replaced with selection medium (25 ug mycophenolic acid (MPA) (Sigma-Aldrich, USA) and 250 ug xanthine (Sigma-Aldrich, USA) per ml of 4% FCS in DMEM/Hams-F12 (Gibco, USA)). The cells were cultured for 72 hours after which a lysate was prepared for FBT cell infection and further culture (72 hours) in selection medium for 3 passages. Serial 10-fold dilutions of the final cell lysate were used to infect 6-well plates of confluent FBT cells and blue foci, detected by standard Gus staining, were picked 72h later. Virus was released by freeze/thawing and the process of infection and purification by focus picking was performed eight times. A stock of rLSDV-grttn was prepared from infected FBT cells, which were lysed 3 days p.i. Following a low-speed centrifugation step to pellet cell debris, the virus was pelleted through a 36% sucrose cushion and resuspended in PBS. MDBK cells were used to determine the virus titre by staining with sheep anti-RT (ARP428) and rabbit anti-sheep HRP (Dako, Denmark). Infected cells were visualized by adding o-Dianisidine (Sigma-Aldrich, USA) and hydrogen peroxide (Sigma-Aldrich, USA).
PCR was used to confirm the absence of wild-type LSDV in the rLSDV-grttn preparation. Three primers were designed: SQRR1, 5'-GTGGGCGTCAATGTTGAC-3' binds specifically to a sequence immediately upstream of the ribonucleotide reductase gene (insertion site). SQGrttn3, 5'-GCTACTTCCCCGACTGGC-3' binds specifically to sequences within and towards the 3' end of the Grttn gene. Anti-sense primer SQRR2, 5'-CATAAAATCAGTACATGCATCC-3' binds specifically to a sequence immediately downstream of the ribonucleotide reductase gene. Confluent monolayers of FBT cells were infected with rLSDV-grttn or wild-type LSDV at a m.o.i of 0.05 pfu per cell. 48 hours post infection lysates were prepared as described by . PCR was performed, using Pfu polymerase (Promega, USA), with an initial denaturation step of 95°C for 2 minutes, followed by 35 cycles of 95°C for 1 minute, 55°C for 30 seconds and 73°C for 5 minutes; the final elongation step was at 73°C for 7 minutes.
FBT cells, grown on glass cover slips, were infected with LSDV and/or transfected with pYS-05, and fixed after 24 hours with methanol-acetone (1:1). Grttn protein was detected with sheep anti-p24 (Aalto Bioreagents Ltd., Ireland) and FITC conjugated donkey anti-sheep (Dako, Denmark) and viewed using a Zeiss fluorescence microscope.
Western blot Analysis to detect protein expression
Cell lysates were prepared 24 h after infection of FBT cells with wild type LSDV (wt LSDV)or LSDVgrttn. For detection of transient expression some wells were transfected with pYS-05 follwing infection with wtLSDV. Proteins were separated using 12% SDS polyacrylamide gel electrophoreses and transferred to a nitrocellulose membrane (Amersham Hybond™-P) using a semi-dry blotting apparatus (Bio-Rad Laboratories, USA). Grttn protein was detected by probing with sheep anti-RT (ARP428) followed by anti-sheep IgG antibody conjugated with alkaline phosphatase and visualized with NBT/BCIP (Roche, Germany).
BALB/c mice (5 mice per group) were used for immune response evaluation. Wild type LSDV (106 ffu or 104 ffu in 100 μl PBS), rLSDV-grttn (106 ffu in 100 μl PBS), rMVA-grttn (106 plaque forming units (pfu) in 100 μl PBS) or pVRC-grttn (100 μg DNA in 100 μl PBS), was administered by the intramuscular route, with 50 μl injected into each quadricep muscle according to approval of the UCT Animal Research Ethics Committee.
IFN-γ and IL-2 ELISPOT assays
IFN-γ and IL-2 Gag- and RT- specific CD8+ and CD4+ T cells were detected using ELISPOT sets (BD Pharmingen) and splenocytes (pooled from 5 mice per group) after red blood cell lysis [42, 43]. Triplicate reactions contained splenocytes (500 000 per well) in 200 μl R10 culture medium (RPMI with 10% heat inactivated FCS, (Gibco, USA) containing 15 mM β-mercaptoethanol, 100 U penicillin per ml, and 100 μg streptomycin). Peptides (>95% pure; Bachem, Switzerland) AMQMLKDTI (GagCD8), NPPIPVGRIYKRWIILGLNK (GagCD4), VYYDPSKDLIA (RTCD8) and PKVKQWPLTEVKIKALTAI (RTCD4) were used at 4 μg/ml. Reactions without peptide were included to determine background responses. Peptide responses are reported as spot-forming units (sfu) per 106 splenocytes after subtraction of background responses. Cumulative ELISPOT responses to Gag and RT were calculated as the sum of responses to the individual CD8+ and CD4+ T cell peptides.
Quantification of antigen-specific cytokine production
Splenocytes (7.5 × 106 per ml R10) were cultured for 24 hours with Gag and RT CD8 and CD4 peptides as used in the IFN-γ ELISPOT assay. IFN-γ in the culture supernatant was quantified using a flow cytometric bead based assay (BD Pharmingen) and calculated as pg IFN-γ per 106 splenocytes . IFN-γ produced during stimulation with the individual CD8+ and CD4+ T cell peptides is expressed as cumulative IFN-γ produced.
Splenocytes (5 × 106) were labeled for 30 min at 4°C with APC-conjugated pentameric H-2Dk AMQMLKDTI or H-2Dd VYYDPSKDLIA complexes (ProImmune, Oxford) and PerCP-conjugated anti-CD8α (clone 53-6.7, eBiosciences) . Flow cytometry (FACScalibur with CellQuest software (BD Biosciences)) was used to analyze pentameric-positive cells as a percentage of gated CD8+ T cells.
Statistical tests were performed using 2-sample Student's t-test and one-way ANOVA test and p values of <0.05 were considered significant.