Development of a modified serum-free medium for Vero cell cultures: effects of protein hydrolysates, l-glutamine and SITE liquid media supplement on cell growth.

Vero cells have been widely used in the viral vaccine production due to the recommendation of the World Health Organization regarding its safety and non-tumorigenicity. The aim of this study was to describe the development a modified serum-free medium for Vero cell cultures. Two protein hydrolysates (Bacto™ soytone and Bacto™ yeast extract), vitamin C, vitamin B12, SITE liquid media supplement, and recombinant human epidermal growth factor (rEGF) were investigated as serum substitutes. A sequential experiment of fractional factorial and central composite design was applied. A modified serum-free medium obtained (named as SFM01-M) was verified. Contrary to P0, the cell yields obtained at P1, P2, and P3 decreased continuously during the verification experiments indicating that Vero cells could not adapt to SFM01-M as expected according to the empirical mathematical model. To improve cell growth after P0, protein hydrolysates, l-glutamine, and SITE liquid media supplement were further investigated. The results showed that cell yields gradually decreased from P1 to P3 when a fixed concentration of Bacto™ yeast extract (7.0 g/L) combined with various concentrations of Bacto™ soytone (0.1-7.0 g/L) in SFM01-M were used. Similarly, cell yields also gradually decreased from P1 to P3 when a fixed concentration of Bacto™ soytone (7.0 g/L) combined with various concentrations of Bacto™ yeast extract (0.1-7.0 g/L) in SFM01-M were used. However, the combination of Bacto™ soytone at 0.1 g/L and Bacto™ yeast extract at 7.0 g/L or Bacto™ soytone at 7.0 g/L and Bacto™ yeast extract at 0.1 g/L in SFM01-M could give the maximum cell yield at P3 when compared with other combinations. In addition, the addition of SITE liquid media supplement (0.1-2.0% v/v) in SFM01-M in which the concentrations of Bacto™ soytone, Bacto™ yeast extract, and l-glutamine were fixed at 0.1 g/L, 0.1 g/L, and 4.0 mM, respectively, the results showed that the cell yields obtained at P3 were not significantly different. From this study, the optimum concentrations of SFM01-M components were as follows: Bacto™ soytone (0.1 g/L), Bacto™ yeast extract (0.1 g/L), vitamin C (9.719 mg/L), vitamin B12 (0.1725 mg/L), SITE liquid media supplement (0.1-2.0% v/v), rEGF (0.05756 mg/L), l-glutamine (4.0 mM), MEM non-essential amino acids (1.0% v/v), sodium pyruvate (1.0 mM), MEM (9.4 g/L), and sodium hydrogen carbonate (2.2 g/L). However, to evaluate SFM01-M in the long-term subculture of Vero cells, the efficiency of SFM01-M will be further investigated.

Molecular evaluation of extracellular activity of medicinal herb Clinacanthus nutans against herpes simplex virus type-2.

Clinacanthus nutans (Burm. f.) Lindau (C. nutans), a medicinal herb belonging to the family Acanthaceae, has traditionally been used in herpes simplex virus (HSV) treatment in Thailand. Clinical trials have indicated that topical preparations produced from its extracts were effective in HSV-2 treatment. However, there is no clear evidence of the mechanism of action or a molecular target of C. nutans. In this study, the extracellular activity of C. nutans extracts against HSV-2 infected on HEp-2 cells was investigated in terms of its molecular aspects. HSV-2 was treated with the extracts and adsorped into the HEp-2 cells. After infection, HSV-2 DNA quantities in the infected cells were assessed and compared by the quantitative dot blot hybridisation technique. The results showed that treating the viruses with either less or more highly purified extracts before infection resulted in great reductions of viral infectivity. Further investigation was performed by Western blot analysis to determine the activities of the extracts on the viral proteins. At least eight viral proteins of the infected cell proteins (ICP) and some structural proteins, including 146, 125, 78, 69, 55, 44, 40 and 20 KDa proteins, were depleted and reduced gradually with higher and lower concentrated herb extracts, respectively. These suggest that the C. nutans extracts highly inactivated or inhibited HSV-2 before infection.

Prime-boost immunization of codon optimized HIV-1 CRF01_AE Gag in BCG with recombinant vaccinia virus elicits MHC class I and II immune responses in mice.

The HIV-1 CRF01_AE gag gene was modified by codon restriction for Mycobacterium spp. and transformed into BCG; and it was designated as rBCG/codon optimized gagE. This produced 11 fold higher HIV-1 gag protein expression than the recombinant native gene rBCG/HIV-1gagE. In mice, CTL activity could be induced either by a single immunization of the codon optimized construct or by using it as a priming antigen in the prime-boost modality with recombinant Vaccinia virus expressing native HIV-1 gag. Specific secreted cytokine responses were also investigated. Only when rBCG gag was codon optimized did the prime-boost immunization produce significantly enhanced IFN-gamma and IL-2 secretion indicating recognition via CD4+ and CD8+ T cells, and these responses seemed to be codon optimized immunogen dose-responsive. On contrary, the prime-boost vaccination using an equal amount of native rBCG/HIV-1gagE instead, or a single rBCG/codon optimized gagE immunization, had no similar effect on the cytokine secretion. These findings suggest that the use of recombinant codon BCG construct with recombinant Vaccinia virus encoding CRF01_AE gag as the prime-boost HIV vaccine candidate, will induce CD4+ Th1 and CD8+ T cell cytokine secretions in addition to enhancing CD8+ CTL response.

Enhancement of cell-mediated immune response in mice by whole HIV-1 gag in Mycobacterium bovis BCG as a live vaccine candidate.

In this study, we employed a recombinant Mycobacterium bovis Bacille Calmette-Guerin (BCG) harboring whole HIV-1 CRF01_AE gag DNA as a candidate vaccine to investigate specific cell-mediated immunity in BALB/c mice. Construction of the stable expression recombinant BCG was achieved by demonstrating by Western blot detection of protein of approximately 55 kDa. By a single injection of 0.1 mg of the recombinant HIV-1 gag protein expressing BCG subcutaneously into mice, after 2 weeks various specific cytotoxic T-lymphocyte (CTL) responses were exhibited against a single gag epitope of amino acid positions 294-304, and also against various peptide regions along the entire gag protein with moderate CTL activities (10-35% specific cell lysis), which increased to relatively high levels (50-68%) after one month. However, after two months activities were 3-3.7 fold lower. On the other hand, gag-specific lymphocyte proliferation was detected 9.3 fold higher than that of non-immunized mouse spleen cells. Our results indicate that in mice, BCG can be used as a recombinant live vector to induce cellular immune responses to HIV-1 gag antigen.

Prime-boost vaccination using recombinant Mycobacterium bovis BCG and recombinant vaccinia virus DIs harboring HIV-1 CRF01_AE gag in mice: influence of immunization routes.

We have previously reported that live vector-based HIV-1 gag vaccine candidate using BCG as a vector was achievable in BALB/c mice. Although the gag-specific CTL induced by this live candidate vaccine is significantly high, persistence of CTL remains unclear. Thus, efforts were made to explore the potential of recombinant Vaccinia virus DIs strain harboring the same HIV-1 CRF01_AE gag gene (rVaccinia/ HIV-1gagE) present in the BCG construct, using different immunization routes. After one month following a single subcutaneous (s.c.) injection of rBCG/HIV-1gagE, higher CTL responses were recognized against various peptide epitopes along the whole gag protein compared to that by intradermal (i.d.) route. A prime-boost regimen having only rDIs/HIV-1gagE injected i.d. induced very low CTL levels. However, within two months, by priming with rBCG/HIV-1gagE s.c. and boosting with rVaccinia/HIV-1gagE intravenously (i.v.), CTL levels were greater (20-68% specific cell lysis) than those obtained by priming and boosting both i.d. (18-35%). After seven months, both prime-boost immunization with rBCG/HIV-lgagE s.c. and with rVaccinia/HIV-1gagE either i.v. or i.d. sustained similar CTL levels. Our studies exhibit that the prime-boost vaccination of rBCG/HIV-1gagE following by rVaccinia/HIV-1gagE i.d. could be used to elicit prolonged CTL responses as well as memory T-cells in mice, which might be more practical than using i.v. route.

Specific immune response and pathological findings in BALB/c mice inoculated with recombinant BCG expressing HIV-1 antigen.

Recombinant BCGs (rBCGs) containing extrachromosomal plasmids with different HIV-1 insert sequences: nef, env (V3J1 and E9Q), gag p17 or whole gag p55 were evaluated for their immunogenicity, safety and persistent infection in BALB/c mice. Animal injected with, rBCG-plJKV3J1, rBCG-pSO gag p17 or rBCG-pSO gag p55 could elicit lymphocyte proliferation as tested by specific HIV-1 peptides or protein antigen. Inoculation with various concentration of rBCG-pSO gag p55 generated satisfactory specific lymphocyte proliferation in dose escalation trials. The rBCG-pSO gag p55 recovered from spleen tissues at different time interval post-inoculation could express the HIV protein as determined by ELISA p24 antigen detection kit. This result indicated that the extrachromosomal plasmid was stable and capable to express Gag protein. It was also demonstrated that rBCGs did not cause serious pathological change in the inoculated animals. The present study suggested the role of BCG as a potential vehicle for using in HIV vaccine development.