ABSTRACT
We have investigated novel vaccines strategies against severe acute respiratory syndrome (SARS) CoV infection using cDNA constructs encoding the structural antigens; spike (S), membrane (M), envelope (E), or nucleocapsid (N) protein, derived from SARS CoV (strain HKU39849, TW1, or FFM-1). As SARS-CoV is thought to infect the alveolar epithelial cell of the lung,in the present study, a type II alveolar epithelial cell clone, T7, was used to analyze the mechanism of CTL against SARS CoV membrane antigens. Mice vaccinated with SARS CoV (N) DNA or (M) DNA using pcDNA 3.1 (+) plasmid vector showed T-cell immune responses (CTL induction and proliferation) against type II alveolar epithelial cells (T7) transfected with SARS (N) or (M) DNA, respectively. To determine whether these DNA vaccines could induce T-cell immune responses in humans as well as in mice, SCID-PBL/hu mice were immunized with these DNA vaccines. PBL from healthy human volunteers were administered i.p. into IL-2 receptor gamma-chain-disrupted NOD-SCID mice [IL-2R(-/-) NOD-SCID]. SCID-PBL/hu mice thus constructed can be used to analyze the human immune response in vivo. The SCID-PBL/hu mice were immunized with SARS (N) DNA or (M) DNA and analyzed for a human T-cell immune response. The M DNA vaccine enhanced CTL activity and proliferation in the presence of M peptide in SCID-PBL/hu mice. Furthermore, the SARS N DNA vaccine induced CTL activity (IFN-gamma production by recombinant N protein or N protein-pulsed autologous B blast cells) and proliferation of spleen cells in SCID-PBL/hu mice. These results, demonstrate that SARS M and N DNA vaccines induced human CTL and human T-cell proliferative responses. On the other hand, we have developed SARS DNA vaccines that induce human neutralizing antibodies and human monoclonal antibodies against SARS CoV. Transgenic mice expressing SARS-CoV receptor (angiotensin converting enzyme 2) are also under development. These vaccines are expected to induce immune responses specific for SARS CoV in human and should provide useful tool for development of protective vaccines.
Subject(s)
Immunization, Passive/methods , Severe Acute Respiratory Syndrome/prevention & control , Severe acute respiratory syndrome-related coronavirus/genetics , Severe acute respiratory syndrome-related coronavirus/metabolism , Viral Vaccines , Animals , Coculture Techniques , Disease Models, Animal , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, SCID , Mice, TransgenicABSTRACT
We investigated the immunogenicity and protective efficacy of DNA vaccine combinations expressing mycobacterial heat shock protein 65 (Hsp65) and interleukin-12 (IL-12) using gene gun bombardment and the hemagglutinating virus of Japan (HVJ)-liposome method. A mouse IL-12 expression vector (mIL-12 DNA) encoding single-chain IL-12 proteins comprised of p40 and p35 subunits were constructed. In a mouse model, a single gene gun vaccination with the combination of Hsp65 DNA and mIL-12 DNA provided a remarkably high degree of protection against challenge with virulent Mycobacterium tuberculosis; bacterial numbers were 100-fold lower in the lungs compared to BCG-vaccinated mice. To explore the clinical use of the DNA vaccines, we evaluated HVJ-liposome encapsulated Hsp65 DNA and mIL-12DNA (Hsp65 + mIL-12/HVJ). The HVJ-liposome method improved the protective efficacy of the Hsp65 DNA vaccine compared to gene gun vaccination. Hsp65 + mIL-12/HVJ induced CD8+ cytotoxic T lymphocyte activity against Hsp65 antigen. Most importantly, Hsp65+mIL-12/HVJ vaccination resulted in a greater degree of protection than that evoked by BCG. This protective efficacy was associated with the emergence of IFN-gamma-secreting T cells and activation of proliferative T cells and cytokines (IFN-gamma and IL-2) production upon stimulation with Hsp65 and antigens from M. tuberculosis. These results suggest that Hsp65 + IL-12/HVJ could be a promising candidate for a new tuberculosis DNA vaccine, which is superior to BCG vaccine.
Subject(s)
Bacterial Proteins/genetics , Chaperonins/genetics , Interleukin-12/genetics , Lymphocyte Activation , Sendai virus/genetics , T-Lymphocytes/immunology , Tuberculosis Vaccines/immunology , Vaccines, DNA/immunology , Animals , Bacterial Proteins/immunology , Biolistics , Chaperonin 60 , Chaperonins/immunology , Female , Granuloma/prevention & control , Interferon-gamma/biosynthesis , Interleukin-12/immunology , Liposomes , Mice , Mice, Inbred BALB C , Tuberculosis Vaccines/administration & dosage , Vaccination , Vaccines, DNA/administration & dosageABSTRACT
We have developed two novel tuberculosis (TB) vaccines: a DNA vaccine combination expressing mycobacterial heat shock protein 65 (Hsp65) and interleukin-12 (IL-12) by using the hemagglutinating virus of Japan (HVJ)-liposome (HSP65+IL-12/HVJ) and a recombinant BCG harboring the 72f fusion gene (72f rBCG). These vaccines provide remarkable protective efficacy in mouse and guinea pig models, as compared to the current by available BCG vaccine. In the present study, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis, to evaluate the HSP65+IL-12/HVJ and 72f rBCG vaccines. Vaccination with HSP65+IL-12/HVJ as well as 72f rBCG vaccines provided better protective efficacy as assessed by the Erythrocyte Sedimentation Rate, chest X-ray findings and immune responses than BCG. Most importantly, HSP65+IL-12/HVJ resulted in an increased survival for over a year. This is the first report of successful DNA vaccination and recombinant BCG vaccination against M. tuberculosis in the monkey model.
Subject(s)
BCG Vaccine/pharmacology , Tuberculosis Vaccines/pharmacology , Tuberculosis, Pulmonary/prevention & control , Animals , BCG Vaccine/genetics , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Chaperonin 60 , Chaperonins/genetics , Chaperonins/immunology , Disease Models, Animal , Guinea Pigs , Humans , Interleukin-12/genetics , Liposomes , Macaca fascicularis , Mice , Sendai virus/genetics , Tuberculosis Vaccines/genetics , Tuberculosis, Pulmonary/immunology , Tuberculosis, Pulmonary/microbiology , Vaccines, DNA/genetics , Vaccines, DNA/pharmacology , Vaccines, Synthetic/genetics , Vaccines, Synthetic/pharmacologyABSTRACT
We have investigated to develop novel vaccines against SARS CoV using cDNA constructs encoding the structural antigen; spike protein (S), membrane protein (M), envelope protein (E), or nucleocapsid (N) protein, derived from SARS CoV. Mice vaccinated with SARS-N or -M DNA using pcDNA 3.1(+) plasmid vector showed T cell immune responses (CTL induction and proliferation) against N or M protein, respectively. CTL responses were also detected to SARS DNA-transfected type II alveolar epithelial cells (T7 cell clone), which are thought to be initial target cells for SARS virus infection in human. To determine whether these DNA vaccines could induce T cell immune responses in humans as well as in mice, SCID-PBL/hu mice was immunized with these DNA vaccines. As expected, virus-specific CTL responses and T cell proliferation were induced from human T cells. SARS-N and SARS-M DNA vaccines and SCID-PBL/hu mouse model will be important in the development of protective vaccines.
