Kiwon Han

Aerosol delivery of spermine-based poly(amino ester)/Akt1 shRNA complexes for lung cancer gene therapy

Polyethylenimine (PEI) has been commonly used as a cationic polymeric gene carrier due to high transfection efficiency, however, its cytotoxicity has hindered the practical application. In this study, we report the development of poly(amino ester) (PAE) based on glycerol propoxylate triacrylate (GPT) and spermine (SPE) as an alternative gene carrier for lung cancer therapy. GPT-SPE copolymer was prepared by Michael addition reaction between GPT and SPE, and the efficacy was evaluated using shAkt1 as a model therapeutic gene. The molecular weight and composition were characterized using gel permeability chromatography (GPC) and (1)H-nuclear magnetic resonance ((1)H-NMR), respectively. The GPT-SPE could effectively condense DNA with about 163 nm size and protect the DNA from nucleases. GPT-SPE/DNA complexes showed excellent transfection with low toxicity both in vitro and in vivo. Furthermore, aerosol delivery of GPT-SPE/Akt1 shRNA complexes significantly suppressed lung tumorigenesis in K-ras(LA1) lung cancer model mice. These results suggest that GPT-SPE can be used in shRNA-based lung cancer gene therapy.

Evaluation of the efficacy of a new modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine (Fostera PRRS) against heterologous PRRSV challenge

The objective of this study was to evaluate a new modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine (Fostera PRRS, Zoetis, Florham, NJ, USA) that was based on a virulent US PRRSV isolate (P129) attenuated using CD163-expressing cell lines. Sixty-four PRRSV-seronegative 3-week-old pigs were randomly divided into the following four groups: vaccinated challenged (group 1), vaccinated unchallenged (group 2), unvaccinated challenged (group 3), and unvaccinated unchallenged (group 4). The pigs in groups 1 and 2 were immunized with a 2.0 mL dose of modified live PRRSV vaccine at 21 days of age, according to the manufacturers recommendations. At 56 days of age (0 days post-challenge), the pigs in groups 1 and 3 were inoculated intranasally with 3 mL of tissue culture fluid containing 10(5) 50% tissue culture infective dose (TCID50)/mL of PRRSV (SNUVR090851 strain, fourth passage in MARC-145 cells). Vaccinated challenged pigs exhibited significantly lower (P<0.05) respiratory scores, viremia, macroscopic and microscopic lung lesion scores, and PRRSV-antigen with interstitial pneumonia than unvaccinated challenged pigs. The induction of PRRSV-specific IFN-γ-SCs by the new modified live PRRSV vaccine produced a protective immune response, leading to the reduction of PRRSV viremia. Although the new modified live PRRSV vaccine is not effective against heterologous PRRSV challenge, the new modified live PRRSV vaccine was able to reduce the levels of viremia and nasal shedding, and severity of PRRSV-induced lesions after challenging virus under experimental conditions.

Effect of the Modified Live Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Vaccine on European and North American PRRSV Shedding in Semen from Infected Boars

ABSTRACT The objective of the present study was to compare the effects of the modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine (Ingelvac PRRS MLV; Boehringer Ingelheim Animal Health, St. Joseph, MO) on European and North American PRRSV shedding in the semen of experimentally infected boars. The boars were randomly divided into six groups. Vaccinated boars shed the North American PRRSV at the rate of 100.1 to 101.0 viral genome copies per ml and 3.63 to 101.1 50% tissue culture infective doses (TCID50)/ml, respectively, in semen, whereas nonvaccinated boars shed the North American PRRSV at the rate of 100.2 to 104.7 viral genome copies per ml and 1.14 to 103.07 TCID50/ml, respectively, in semen. Vaccinated boars shed the European PRRSV at the rate of 100.1 to 104.57 viral genome copies per ml and 1.66 to 103.10 TCID50/ml, respectively, in semen, whereas nonvaccinated boars shed the European PRRSV at the rate of 100.3 to 105.14 viral genome copies per ml and 1.69 to 103.17 TCID50/ml, respectively, in semen. The number of genomic copies of the European PRRSV in semen samples was not significantly different between vaccinated and nonvaccinated challenged European PRRSV boars. The present study demonstrated that boar vaccination using commercial modified live PRRSV vaccine was able to decrease subsequent shedding of North American PRRSV in semen after challenge but was unable to decrease shedding of European PRRSV in semen after challenge.

GOLGA2/GM130, cis-Golgi matrix protein, is a novel target of anticancer gene therapy.

Achievement of long-term survival of patients with lung cancer treated with conventional chemotherapy is still difficult for treatment of metastatic and advanced tumors. Despite recent progress in investigational therapies, survival rates are still disappointingly low and novel adjuvant and systemic therapies are urgently needed. A recently elucidated secretory pathway is attracting considerable interest as a promising anticancer target. The cis-Golgi matrix protein, GOLGA2/GM130, plays an important role in glycosylation and transport of protein in the secretory pathway. In this study, the effects of short hairpin RNA (shRNA) constructs targeting GOLGA2/GM130 (shGOLGA2) on autophagy and lung cancer growth were evaluated in vitro and in vivo. Downregulation of GOLGA2/GM130 led to induction of autophagy and inhibition of glycosylation in A549 cells and in the lungs of K-ras(LA1) mice. Furthermore, downregulation of GOLGA2/GM130 decreased angiogenesis and cancer cell invasion in vitro and suppressed tumorigenesis in lung cancer mice model. The tumor specificity of sequence targeting GOLGA2/GM130 was also demonstrated. Taken together, these results suggest that induction of autophagy by shGOLGA2 may induce cell death rather than cell survival. Therefore, downregulation of GOLGA2/GM130 may be a potential therapeutic option for lung cancer.

Efficacy of a reformulated inactivated chimeric PCV1-2 vaccine based on clinical, virological, pathological and immunological examination under field conditions.

Inactivated chimeric porcine circovirus (PCV) 1-2 vaccine was initially taken off the market due to concerns that the vaccine virus was not killed and thus further replicated and spread in the pig population. In August 2011, a reformulated inactivated chimeric PCV1-2 vaccine re-entered the market. The efficacy of the reformulated inactivated chimeric PCV1-2 vaccine was evaluated under field conditions for registration as recommended by the Republic of Koreas Animal, Plant & Fisheries Quarantine & Inspection Agency. Three farms were selected based on their history of postweaning multisystemic wasting syndrome (PMWS). On each farm, a total of 50 3-week-old pigs were randomly allocated to one of two treatment groups: (i) vaccinated at 3 weeks of age and (ii) non-vaccinated. Clinical examination indicated that vaccinated animals displayed an improved average daily weight gain (672.2g/day vs. 625g/day; difference of +47.3g/day; P<0.05) and a reduced time to market (177 days vs. 183 days; difference of -6 days; P<0.05). Virological examination indicated that vaccinated animals displayed a reduced PCV2 load in the blood and nasal swabs compared to non-vaccinated animals. Pathological examination indicated that vaccination of pigs against PCV2 effectively reduced the number of PMWS-associated microscopic lesions and the PCV2 load in lymphoid tissues compared to non-vaccinated animals in the 3 herds. Immunological examination indicated that vaccinated animals induced PCV2-specific neutralizing antibodies (NA) and interferon-γ-secreting cells (IFN-γ-SCs). A reduction in the PCV2 load in the blood coincided with the appearance of both PCV2-specific NA and IFN-γ-SCs in the vaccinated animals. The number of CD4(+) cells was decreased in non-vaccinated animals compared to vaccinated animals. The reformulated inactivated chimeric PCV1-2 vaccine seems to be very effective in controlling PCV2 infection based on clinical, virological, pathological, and immunological evaluations under field conditions.

Protective effect of the maternally derived porcine circovirus type 2 (PCV2)-specific cellular immune response in piglets by dam vaccination against PCV2 challenge.

The objective of the present study was to evaluate (i) the passive transfer of maternally derived functional porcine circovirus type 2 (PCV2)-specific lymphocytes of seronegative sows immunized with the PCV2 vaccine to newborn piglets and (ii) the functional role of the maternally derived PCV2-specific cellular immune response in protecting newborn piglets from challenge with PCV2. After ingesting colostrums, piglets from vaccinated sows (PT01 and PT02) have significantly higher numbers of PCV2-specific gamma interferon-secreting cells, an increased PCV2-specific delayed type hypersensitivity response, and a stronger proliferative response of peripheral blood mononuclear cells compared with piglets from non-vaccinated seronegative sows (PT03 and PT04). In the PCV2 challenge study, the number of serum genomic PCV2 copies was significantly less in piglets from vaccinated sows (PT02) compared with piglets from non-vaccinated sows (PT04) at 7-28 days post-inoculation (P<0.05 and P<0.001). The histopathological lesions and immunohistochemical scores were significantly lower in piglets of vaccinated sows compared with those of non-vaccinated sows. To our knowledge, this is the first report of transferring a maternally derived PCV2-specific cellular immune response from vaccinated dams to their offspring. Maternally derived adaptive cellular immune responses play a critical role in protecting newborn piglets challenged with PCV2 at 3 weeks of age.

Vaccination of sows against type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) before artificial insemination protects against type 2 PRRSV challenge but does not protect against type 1 PRRSV challenge in late gestation

The objective of the present study was to determine the effects of the commercially available type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)-based modified live vaccine against type 1 and type 2 PRRSV challenge in pregnant sows. Half of the sows in the study were vaccinated with a type 2 PRRSV-based vaccine 4 weeks prior to artificial insemination while the other half remained non-vaccinated. Sows were then challenged intranasally with type 1 or type 2 PRRSV at 93 days of gestation. The sows which received the type 2 PRRSV-based vaccine followed by type 2 PRRSV challenge had significantly higher neutralizing antibody titers against type 2 PRRSV than they did against type 1 PRRSV. These same sows had higher frequencies of IFN-γ-secreting cells when stimulated with type 2 PRRSV compared to those stimulated with type 1 PRRSV. Subsequent virological evaluation demonstrated that the type 2 PRRSV-based vaccine reduced the type 2 PRRSV load but not the type 1 PRRSV load present in the blood of the sows. Additionally, vaccination of pregnant sows with the type 2 PRRSV-based vaccine effectively reduced the level of type 2 PRRSV nucleic acids observed in fetal tissues from type 2 PRRSV-challenged sows but did not reduce the level of type 1 PRRSV nucleic acid observed in fetal tissues from type 1 PRRSV-challenged sows. This study demonstrates that the vaccination of pregnant sows with the type 2 PRRSV-based vaccine protects against type 2 PRRSV challenge but does not protect against type 1 PRRSV challenge.

Reduction of porcine circovirus type 2 (PCV2) viremia by a reformulated inactivated chimeric PCV1-2 vaccine-induced humoral and cellular immunity after experimental PCV2 challenge

BackgroundThe objective of the present study was to elucidate the humoral and cellular immune response mechanisms by which a reformulated inactivated chimeric PCV1-2 vaccine reduces the PCV2 viremia. Forty PCV2 seronegative 3-week-old pigs were randomly divided into the following four groups: vaccinated challenged (T01), vaccinated non-challenged (T02), non-vaccinated challenged (T03), and non-vaccinated non-challenged (T04) animals. The pigs in groups T01 and T02 were immunized with a reformulated inactivated chimeric PCV1-2 vaccine (Fostera™ PCV; Pfizer Animal Health) administered as a 2.0 ml dose at 21 days of age. At 35 days of age (0 days post-challenge), the pigs in groups T01 and T03 were inoculated intranasally with 2 ml each of PCV2b.ResultsA reduction of PCV2 viremia coincided with the appearance of both PCV2-specific neutralizing antibodies (NA) and interferon-γ-secreting cells (IFN-γ-SCs) in the vaccinated animals. However, the presence of anti-PCV2 IgG antibodies did not correlate with the reduction of PCV2 viremia. Lymphocyte subset analysis indicated that the numbers of CD3+ and CD4+ cells increased in vaccinated animals but the numbers of CD4+ cells decreased transiently in non-vaccinated animals. The observation of a delayed type hypersensitivity response in only the vaccinated animals also supports a CD4+ cell-associated protective cellular immune response induced by the reformulated inactivated chimeric PCV1-2 vaccine.ConclusionsThe induction of PCV2-specific NA and IFN-γ-SCs, and CD4+ cells by the reformulated inactivated chimeric PCV1-2 vaccine is the important protective immune response leading to reduction of the PCV2 viremia and control of the PCV2 infection. To our knowledge this is the first demonstration of protective humoral and cellular immunity induced by the reformulated inactivated chimeric PCV1-2 vaccine and its effect on reduction of PCV2 viremia by vaccination.

Comparative Effects of Vaccination against Porcine Circovirus Type 2 (PCV2) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in a PCV2-PRRSV Challenge Model

ABSTRACT The objective of the present study was to determine the effects of porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) vaccinations in an experimental PCV2-PRRSV challenge model, based on virological (viremia), immunological (neutralizing antibodies [NAs], gamma interferon-secreting cells [IFN-γ-SCs], and CD4+ CD8+ double-positive cells), and pathological (lesions and antigens in lymph nodes and lungs) evaluations. A total of 72 pigs were randomly divided into 9 groups (8 pigs per group): 5 vaccinated and challenged groups, 3 nonvaccinated and challenged groups, and a negative-control group. Vaccination against PCV2 induced immunological responses (NAs and PCV2-specific IFN-γ-SCs) and reduced PCV2 viremia, PCV2-induced lesions, and PCV2 antigens in the dually infected pigs. However, vaccination against PCV2 did not affect the PRRSV immunological responses (NAs and PRRSV-specific IFN-γ-SCs), PRRSV viremia, PRRSV-induced lesions, or PRRSV antigens in the dually infected pigs. Vaccination against PRRSV did not induce immunological responses (PRRSV-specific IFN-γ-SCs) or reduce PRRSV viremia, PRRSV-induced lesions, or PRRSV antigens in the dually infected pigs. In addition, vaccination against PRRSV increased PCV2 viremia, PCV2-induced lesions, and PCV2 antigens in the dually infected pigs. In summary, vaccination against PCV2 reduced PCV2 viremia, PCV2-induced lesions, and PCV2 antigens in the dually infected pigs. However, vaccination against PRRSV increased PCV2 viremia, PCV2-induced lesions, and PCV2 antigens in the dually infected pigs. Therefore, the PCV2 vaccine decreased the potentiation of PCV2-induced lesions by PRRSV in dually infected pigs. In contrast, the PRRSV vaccine alone did not decrease the potentiation of PCV2-induced lesions by PRRSV in dually infected pigs.

Clinical, virological, immunological and pathological evaluation of four porcine circovirus type 2 vaccines.

The objective of this study was to rigorously compare the efficacy of four porcine circovirus type 2 (PCV2) vaccines of varying antigen type and dose under experimental conditions based on well-defined clinical (average daily weight gain [ADWG]), virological (evidence of viraemia), immunological (presence of PCV2-specific neutralising antibodies [NA], interferon-γ-secreting cells [IFN-γ-SCs], and CD3(+) and CD4(+) T cell subsets), and pathological (lymphoid lesion and PCV2 antigen score) criteria. A total of 60, 3-week old piglets were assigned to six groups of 10/group and were vaccinated either with 1/4 commercially available one-dose vaccines or were not vaccinated. At 7 weeks of age, vaccinated and control animals were inoculated intranasally with 2 mL of PCV2b. All pigs were euthanased and subjected to post-mortem examination at 25 weeks of age. From 9 to 16 weeks of age, the ADWG of vaccinated animals was significantly higher than that of non-vaccinates. Significant (P<0.05) differences were observed between vaccinated and positive control groups in the quantity of log-transformed PCV2b DNA in the blood and nasal swabs, log-transformed NA titres, and PCV2-specific IFN-γ-SCs at 0, 7, 14, 21, and 42 days post challenge (dpc). The proportion of CD4(+) cells at 7 and 14 dpc was also significantly different between vaccinated and control pigs (P<0.05). The histopathological lesions and PCV2-antigen scores in the lymph nodes were significantly lower (P<0.05) in vaccinated animals. All four vaccines were found to be highly efficacious in controlling experimental PCV2 challenge based on this range of criteria.