Assessment of the acid phosphatase CP01850 from Corynebacterium pseudotuberculosis in DNA and subunit vaccine formulations against caseous lymphadenitis / Avaliação da fosfatase ácida recombinante CP01850 de Corynebacterium pseudotuberculosis em formulações vacinais de subunidade e de DNA contra a linfadenite caseosa

The target cp1002_RS01850 from Corynebacterium pseudotuberculosis was used to construct a DNA and recombinant subunit vaccine against caseous lymphadenitis. Recombinant protein rCP01850 was expressed in Escherichia coli using pAE vector, and DNA vaccine was engineered with pTARGET vector. BALB/c mice were divided in five groups containing eight animals each, inoculated with: pTARGET/cp01850 as DNA vaccine (G1); rCP01850 plus Al (OH)3 as recombinant subunit vaccine (G2); pTARGET/cp01850 and a boost with rCP01850 plus Al (OH)3 (G3); pTARGET (G4); or Al (OH)3 (G5). Mice were inoculated and blood samples were collected on days 0, 21, and 42 for the analysis of total IgG, IgG1 and IgG2a by ELISA. In each group, five animals were challenged with Mic-6 C. pseudotuberculosis strain, and three were used for cytokine quantification by qPCR. Although no group has been protected by vaccines against lethal challenge, G2 showed an increase in the survival rate after challenge. Significantly higher levels of IL-4, IL-12, IFN-γ, total IgG, IgG1 and IgG2a were also detected for G2, evidencing a mixed Th1/Th2 immunological profile. In conclusion, despite no protection level provided by different vaccinal strategies using cp1002_RS01850 from C. pseudotuberculosis, G2 developed a Th1/Th2 immune response with an increase in survival rate.(AU)

O alvo cp1002_RS01850 de Corynebacterium pseudotuberculosis foi utilizado para construir uma vacina recombinante de subunidade e de DNA contra a linfadenite caseosa. A proteína recombinante rCP01850 foi expressa em Escherichia coli usando o vetor pAE, e a vacina de DNA foi construída com o vetor pTARGET. Camundongos BALB/c foram divididos em grupos de oito animais, inoculados com: pTARGET/cp01850 como vacina de DNA (G1); rCP01850 e Al (OH)3 como vacina recombinante de subunidade (G2); pTARGET/cp01850 e um boost com rCP01850 e Al (OH)3 (G3); pTARGET (G4); ou Al (OH)3 (G5). Os animais foram inoculados e amostras de sangue foram coletadas nos dias 0, 21, e 42 do experimento para a análise de IgG total, IgG1 e IgG2a por ELISA. De cada grupo, cinco animais foram desafiados com a cepa Mic-6 de C. pseudotuberculosis, e três foram usados para a quantificação de citocinas por qPCR. Apesar de nenhum grupo ter sido protegido pelas vacinas testadas contra o desafio letal, G2 apresentou taxa de sobrevida e níveis de IL-4, IL-12, IFN-γ, IgG total, IgG1 e IgG2a significativamente mais altos, evidenciando um perfil imunológico misto Th1/Th2. Conclui-se que apesar das diferentes estratégias vacinais utilizando cp1002_RS01850 de C. pseudotuberculosis não terem sido capazes de gerar proteção, G2 desenvolveu uma resposta Th1/Th2 e elevou a taxa de sobrevida.(AU)

Immune responses induced by BCG prime and single dose of recombinant adenovirus Ad5-CEAB boosted strategy in mice / 中国人兽共患病学报

Tuberculosis (TB) remains an enormous health burden worldwide.To date,Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the unique anti-TB vaccine available for humans,which provides an important but limited protection from the Mycobacterium tuberculosis (Mtb) infection.It is therefore an urgent need to develop better vaccines and vaccination strategies to prevent the spread of Mtb infection.Heterologous prime-boost vaccination strategies using both BCG and novel anti-TB vaccines have been demonstrated to induce robust immune responses than BCG alone.We have previously demonstrated that a recombinant adenoviral vector Ad5-CEAB co-expressing CFP10,ESAT6,Ag85A and Ag85B of Mtb was able to induce robust antigen-specific immune responses in mice.In the present study,we examined immunological effects of Ad5-CEAB in the mice primed with BCG and boosted with a single dose of the virus via an intranasal route.Results demonstrated that this vaccination strategy could effectively induce strong antigen-specific mucosal and humoral immune responses.These immune responses were characterized with an increased productions of cytokines (IL-12 and IFN-γ),increased concentration of secretary IgA (sIgA) in bronchoalveolar lavage fluid (BALF) and serum IgG in mice in comparison with mice in BCG group.These data suggested that the regimen of BCG prime-single dose of Ad5-CEAB boosted strategy was novel for inducing antigen-specific immune responses in response to Mtb antigens in vivo,which warrants for further development of adenoviral-based vaccine against Mtb infection.

Cross protective immune responses in mice elicited by prime-boost strategy with a recombinant DNA vaccine and adenoviral 5-based vaccine expressing structural antigens of hepatitis C virus / 中华微生物学和免疫学杂志

Objective To investigate the development strategy of novel T cell based vaccine against HCV infection.Methods BALB/c mice were primed with pSCK-based DNA vaccine and boosted with type 5 adenoviral vector-based vaccine, which expressed the structural proteins ( Core, E1 and E2) de-rived from a Chinese HCV patient (genotype 1b, Hebei strain).Enzyme linked immunospot assay (ELIS-POT) and intracellular cytokine staining ( ICS) were used to analyze the elicited antigen-specific immune re-sponses and the efficacy of cross-protection.Results Immunization of mice with the prime-boost vaccination strategy elicited stronger T cell immune responses against multiple HCV antigens than using the DNA vac-cines alone, especially the IFN-γ-secreting T cell responses against E1 protein as indicated by ELISPOT as-say.ICS data indicated that the prime-boost regimen elicited more TNF-α-producing CD4+and IFN-γ-produ-cing CD8+T cells against E1 protein and high levels of IFN-γ-producing CD4+and CD8+T cells against E2 protein in comparison with immunization with DNA vaccines.Moreover, the prime-boost vaccination was ca-pable of eliciting effective cross-protection in a surrogate challenge model based on a recombinant heterolo-gous HCV (JFH1, 2a) vaccinia virus.Conclusion The prime-boost vaccination using DNA and rAd5-based vaccine expressing HCV structural antigens induced significant cellular immune response and cross-protection in mice, suggesting the possibility of using it as a promising T cell based vaccine against HCV in-fection.

DNA prime-protein boost based vaccination with a conserved region of leptospiral immunoglobulin-like A and B proteins enhances protection against leptospirosis

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of theLeptospira genus. Vaccination with bacterins has severe limitations. Here, we evaluated the N-terminal region of the leptospiral immunoglobulin-like B protein (LigBrep) as a vaccine candidate against leptospirosis using immunisation strategies based on DNA prime-protein boost, DNA vaccine, and subunit vaccine. Upon challenge with a virulent strain ofLeptospira interrogans, the prime-boost and DNA vaccine approaches induced significant protection in hamsters, as well as a specific IgG antibody response and sterilising immunity. Although vaccination with recombinant fragment of LigBrep also produced a strong antibody response, it was not immunoprotective. These results highlight the potential of LigBrep as a candidate antigen for an effective vaccine against leptospirosis and emphasise the use of the DNA prime-protein boost as an important strategy for vaccine development.

Effects of various prime-boost regimes on immunities in mice by using DNA and reocmbinant vaccin-ia-based H5N1 vaccines / 中华微生物学和免疫学杂志

Objective To develop an effective and broad immune protective vaccination strategy by using DNA and recombinant vaccinia-based H5N1 vaccines.Methods BALB/c mice were immunized with various prime-boost regimens by using different DNA ( pIRES-based or pVRC-based) and recombinant vaccinia (Tiantan strain, rTTV)-based H5N1 vaccines expressing multivalent antigens (HA, NA, M1 and M2).The differences of immunity induced by two DNA vaccines were compared between intradermal electro -poration (IDE) and intramuscular electroporation (IME) deliveries.Immune responses were analyzed by hemagglutination inhibition( HAI) assay, neuraminidase ( NA)-specific antibody measured by ELISA , mi-croneutralization assay and IFN-γELISPOT assay .Results High levels of humoral immunity and T cell re -sponses were induced in mice primed with DNA-based vaccine than those primed with rTTVb-ased vaccine . DNA priming by IDE resulted in higher levels of neutralizing antibody in mice than those by IME delivery . Higher levels of HAI and anti-NA antibodies as well as NA-specific T cell responses were induced by pVRC-based DNA prime than those by pIRES-based DNA prime .HA-specific T cell responses were also enhanced in mice primed with pVRC-based DNA than those primed with pIRES-based DNA by IME .Conclusion The prime-boost strategies by using DNA-based vaccine in combination with rTTV-based H5N1 vaccine could induce humoral and T cell responses in mice against multi-antigens .Immunities induced by vaccines in com-bination might be modulated by various prime regimes .The study provided references for the further develop-ment of novel H5N1 vaccine and the optimization of immunization programs of combined multi-antigen vac-cine candidates .

Protection of chickens against infectious bronchitis virus with a multivalent DNA vaccine and boosting with an inactivated vaccine

The protective efficacy of DNA plasmids encoding avian infectious bronchitis virus (IBV) S1, N, or M protein was investigated in chickens. Chickens were inoculated monovalently (with plasmid pVAX1-16S1, pVAX1-16M, or pVAX1-16N alone) or multivalently (combination of the three different plasmids, pVAX1-16S1/M/N). A prime-boost immunization protocol against IBV was developed. Chickens were immunized with the multivalent DNA vaccine twice and then boosted with an inactivated vaccine once. Antibody titers of the chickens immunized with pVAX1-16S1/M/N were much higher than those of the monovalent groups (p < 0.01). A protective rate up to 90% was observed in the pVAX1-16S1/M/N group. The serum antibody titers in the prime-boost birds were significantly higher than those of the multivalent DNA vaccine group (p < 0.01) but not significantly different compared to the inactivated vaccine group at 49 days of age. Additionally, the prime-boost group also showed the highest level of IBV-specific cellular proliferation compared to the monovalent groups (p < 0.01) but no significant difference was found compared to the multivalent DNA vaccine group, and the prime-boost group completely protected from followed viral challenge.

Establishment of a guinea pig model for evaluating the protective effects of new TB vaccines in BCG prime-boost regimen / 中华微生物学和免疫学杂志

Objective To establish a suitable guinea pig model for evaluating the protective effects of new TB vaccines in BCG prime-boost regimen .Methods Two different immunization strategies by using the recombinant TB vaccine were employed to boost BCG primed guinea pigs in this study .One was for short-term evaluation with 14 weeks interval between prime and boost immunization and another was for long -term evaluation with 54 weeks interval .In the short-term evaluation group , guinea pigs were boosted twice with the recombinant TB vaccine ( AEC/BC02 ) in every two weeks , while guinea pigs in the long-term evaluation group were boosted for three times with two weeks interval between each injection .A negative con-trol group ( NS→NS) and a BCG control group ( BCG→NS) were both set up in two evaluation groups .One week after the last immunization , all guinea pigs were challenged with M.tuberculosis.Six to seven weeks after bacteria challenge , all animals were euthanized and dissected to evaluate lesion scores of liver , spleen and lung, as well as the viable bacterial load in spleen .Results In the short-term evaluation group , the le-sion scores in those boosted with vaccine (3.33±5.00) was lower than that of BCG control group (5.56± 7.27) (P>0.05) and negative control group (47.00±28.11) (P=0.0001).The difference between BCG control group and negative control group in lesion score was also significant .The animals in vaccine boosted group had lower bacterial loads (0.78±1.55 log10 ) in spleen than that in BCG control group (1.06±1.87) (P>0.05) and negative control group (5.47±0.61) (P=0.0003).In the long-term evaluation group, the lesion score in those boosted with vaccine was lower (5.0±7.6) than that in BCG control group (14.4± 13.5) (P=0.0394) and negative control group (56.9±14.1) (P0.05) and negative control group (5.43±0.56) (P=0.01).There was a significant difference in bacterial load between BCG control group and negative group (P=0.0089).Conclusion The results suggest that the interval time between BCG-prime and boost immunization should be properly prolonged in the guinea pig model used for evaluating the protective effects of new TB vaccines in BCG prime -boost regimen .

Immune responses induced by DNA vaccine combined with recombinant adenovirus containing MOMP gene of Chlamydia trachomatis serovar E / 中华微生物学和免疫学杂志

Objective To investigate the specific immune responses induced by DNA vaccine com-bined with recombinant adenovirus carrying major outer membrane protein ( MOMP) gene of Chlamydia trachom-atis (Ct) serovar E.Methods Recombinant eukaryotic expression plasmid pVAX 1-MOMP and recombinant adenovirus Ad-MOMP were constructed and purified .Four immunization strategies were designed including DNA immunization (DNA group), recombinant adenovirus immunization (Ad group), DNA prime-recombinant ade-novirus boost regimen ( DNA/Ad group ) and recombinant adenovirus prime-DNA boost regimen ( Ad/DNA group) .Two weeks after the final immunization , vaginal wash specimens , blood samples and spleens were col-lected from all mice for the evaluation of humoral and cellular immune responses .Results Th1 responses and mucosal responses were not found in DNA group .Ad group induced both Th 1 responses and local mucosal re-sponses , and its IgG and IgA levels were significantly higher than those in DNA group .DNA/Ad group and Ad/DNA group induced stronger Th 1 responses and humoral responses than DNA and Ad group .Serum antibodies and mucosal antibodies in DNA/Ad group were higher than those in Ad/DNA group, but Th1 responses in DNA/Ad group were impaired than those in Ad/DNA group.Conclusion The recombinant adenovirus could induce Th1 responses and genital mucosal responses .Combined immunization of DNA/Ad or Ad/DNA showed enhanced specific immune responses in comparison with the single immunization with DNA or Ad .The differ-ences between the two combination methods were that DNA /Ad group stimulated higher levels of specific anti-body responses , while Ad/DNA group produced stronger Th 1 responses .The study provided a reference for the enhancement and development of vaccines against Chlamydia trachomatis in further studies .

Avaliação do potencial protetor da vacina gênica pvaxapa- dmt-me no modelo experimental de tuberculose / Protective potential evaluation of genetic vaccines pvaxapa- dmt-me in the experimental model of tuberculosis

A tuberculose (TB) é responsável por dois milhões de mortes a cada ano, apesar daampla utilização da vacina BCG (Bacilo Calmette-Guérin). Embora essa vacina nãoproteja contra a TB pulmonar no adulto, protege contra as formas graves da TB nainfância. Para muitos autores, a imunização heteróloga (prime-boost) talvezseja uma das estratégias mais importantes e realistas para o controle da TB,principalmente nos países endêmicos. Muitos estudos têm demonstrado que aimunização com BCG seguido de reforço com vacina gênica (genes de M.tuberculosis) induz melhor proteção contra TB do que somente a vacina BCG. Alémdisso, a estratégia prime-boost utilizando microesferas biodegradáveis pareceser uma imunização promissora para estimular a resposta imune de longa duração.A glicoproteína APA foi identificada como importante antígeno imunodominante,induzindo níveis significativos de proteção contra M. tuberculosis. Assim, esteestudo teve como objetivo avaliar a eficácia protetora da imunização heteróloga emmodelo experimental de TB. Para isso, camundongos BALB/c foram imunizados porvia subcutânea com BCG seguido de um reforço por via intramuscular com aformulação vacinal contendo apa e DMT co-encapsuladosem microesferas (BCG/APA). Essa estratégia prime-boost foi eficiente na indução daresposta imune protetora à infecção por M. tuberculosis, caracterizada pela reduçãosignificativa do número de bacilos no pulmão destes animais na fase mais tardia dainfecção (70 dias após desafio). Além de ser significativamente mais imunogênciaque a vacina BCG, permitiu uma melhor preservação do parênquima pulmonar, comredução do número e tamanho dos granulomas, limitando a extensão da injúriatecidual induzida pela infecção e restringindo a inflamação no tecido alveolar. Apotencialização do efeito protetor da vacina BCG induzida pelo reforço com a vacinapVAXapa+DMT-Me sugere que a estratégia utilizada pode ser importante para aprevenção da TB...

Tuberculosis (TB) is responsible for two millions of deaths each year despite thewidespread use of BCG vaccine (Bacillus Calmette-Guérin). Although this vaccinedoes not protect against adult pulmonary TB, it is protective against severe forms ofchildhood TB. For many authors, heterologous prime-boost regimen is perhaps themost realistic strategy for future TB control, especially in endemic countries. Manystudies have demonstrated that BCG-prime DNA-(M. tuberculosis genes)-boosterinduce greater protection against TB than BCG alone. APA glycoprotein has beenidentified as major immunodominant antigen and induces significant levels ofprotection against M. tuberculosis. Moreover, prime-boost strategy by usingbiodegradable microspheres seems to be a promising immunization to stimulate longlasting immune response. Thus, this study had as aim to investigate the immuneprotection against M. tuberculosis challenge in mice based on BCG priming and DNAvaccine boosting. For that, BALB/c mice were inoculated subcutaneously with aheterologous BCG priming-DNA boosting immunization using pVAXapa and TDM coencapsulatedin microspheres (BCG/APA), intramuscularly. This prime-boost strategywas effective in inducing protective immune response against M. tuberculosisinfection, characterized by significant lower numbers of bacilli in the lungs of theseanimals, in the late phase of infection (70 days after infection). In addition to beingsignificantly more immunogenic than BCG, it allowed better preservation of the lungparenchyma and reduction in the number and size of granulomas, limiting the extentof lung injury induced by infection and inflammation in the alveolar tissue. Theimprovement of the protective effect of BCG vaccine mediated by pVAXapa boostersuggests that our strategy can be important in the treatment for prevention of TB...

Distinct Humoral and Cellular Immunity Induced by Alternating Prime-boost Vaccination Using Plasmid DNA and Live Viral Vector Vaccines Expressing the E Protein of Dengue Virus Type 2

BACKGROUND: Dengue virus, which belongs to the Flavivirus genus of the Flaviviridae family, causes fatal dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) with infection risk of 2.5 billion people worldwide. However, approved vaccines are still not available. Here, we explored the immune responses induced by alternating prime-boost vaccination using DNA vaccine, adenovirus, and vaccinia virus expressing E protein of dengue virus type 2 (DenV2). METHODS: Following immunization with DNA vaccine (pDE), adenovirus (rAd-E), and/or vaccinia virus (VV-E) expressing E protein, E protein-specific IgG and its isotypes were determined by conventional ELISA. Intracellular CD154 and cytokine staining was used for enumerating CD4+ T cells specific for E protein. E protein-specific CD8+ T cell responses were evaluated by in vivo CTL killing activity and intracellular IFN-gamma staining. RESULTS: Among three constructs, VV-E induced the most potent IgG responses, Th1-type cytokine production by stimulated CD4+ T cells, and the CD8+ T cell response. Furthermore, when the three constructs were used for alternating prime-boost vaccination, the results revealed a different pattern of CD4+ and CD8+ T cell responses. i) Priming with VV-E induced higher E-specific IgG level but it was decreased rapidly. ii) Strong CD8+ T cell responses specific for E protein were induced when VV-E was used for the priming step, and such CD8+ T cell responses were significantly boosted with pDE. iii) Priming with rAd-E induced stronger CD4+ T cell responses which subsequently boosted with pDE to a greater extent than VV-E and rAd-E. CONCLUSION: These results indicate that priming with live viral vector vaccines could induce different patterns of E protein- specific CD4+ and CD8+ T cell responses which were significantly enhanced by booster vaccination with the DNA vaccine. Therefore, our observation will provide valuable information for the establishment of optimal prime-boost vaccination against DenV.

Distinct Humoral and Cellular Immunity Induced by Alternating Prime-boost Vaccination Using Plasmid DNA and Live Viral Vector Vaccines Expressing the E Protein of Dengue Virus Type 2

BACKGROUND: Dengue virus, which belongs to the Flavivirus genus of the Flaviviridae family, causes fatal dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) with infection risk of 2.5 billion people worldwide. However, approved vaccines are still not available. Here, we explored the immune responses induced by alternating prime-boost vaccination using DNA vaccine, adenovirus, and vaccinia virus expressing E protein of dengue virus type 2 (DenV2). METHODS: Following immunization with DNA vaccine (pDE), adenovirus (rAd-E), and/or vaccinia virus (VV-E) expressing E protein, E protein-specific IgG and its isotypes were determined by conventional ELISA. Intracellular CD154 and cytokine staining was used for enumerating CD4+ T cells specific for E protein. E protein-specific CD8+ T cell responses were evaluated by in vivo CTL killing activity and intracellular IFN-gamma staining. RESULTS: Among three constructs, VV-E induced the most potent IgG responses, Th1-type cytokine production by stimulated CD4+ T cells, and the CD8+ T cell response. Furthermore, when the three constructs were used for alternating prime-boost vaccination, the results revealed a different pattern of CD4+ and CD8+ T cell responses. i) Priming with VV-E induced higher E-specific IgG level but it was decreased rapidly. ii) Strong CD8+ T cell responses specific for E protein were induced when VV-E was used for the priming step, and such CD8+ T cell responses were significantly boosted with pDE. iii) Priming with rAd-E induced stronger CD4+ T cell responses which subsequently boosted with pDE to a greater extent than VV-E and rAd-E. CONCLUSION: These results indicate that priming with live viral vector vaccines could induce different patterns of E protein- specific CD4+ and CD8+ T cell responses which were significantly enhanced by booster vaccination with the DNA vaccine. Therefore, our observation will provide valuable information for the establishment of optimal prime-boost vaccination against DenV.

Leukotrienes are not essential for the efficacy of a heterologous vaccine against Mycobacterium tuberculosis infection

Leukotrienes are reported to be potent proinflammatory mediators that play a role in the development of several inflammatory diseases such as asthma, rheumatoid arthritis and periodontal disease. Leukotrienes have also been associated with protection against infectious diseases. However, the role of leukotrienes in Mycobacterium tuberculosis infection is not understood. To answer this question, we studied the role of leukotrienes in the protective immune response conferred by prime-boost heterologous immunization against tuberculosis. We immunized BALB/c mice (4-11/group) with subcutaneous BCG vaccine (1 x 10(5) M. bovis BCG) (prime) followed by intramuscular DNA-HSP65 vaccine (100 µg) (boost). During the 30 days following the challenge, the animals were treated by gavage daily with MK-886 (5 mg·kg-1·day-1) to inhibit leukotriene synthesis. We showed that MK-886-treated mice were more susceptible to M. tuberculosis infection by counting the number of M. tuberculosis colony-forming units in lungs. The histopathological analysis showed an impaired influx of leukocytes to the lungs of MK-886-treated mice after infection, confirming the involvement of leukotrienes in the protective immune response against experimental tuberculosis. However, prime-boost-immunized mice treated with MK-886 remained protected after challenge with M. tuberculosis, suggesting that leukotrienes are not required for the protective effect elicited by immunization. Protection against M. tuberculosis challenge achieved by prime-boost immunization in the absence of leukotrienes was accompanied by an increase in IL-17 production in the lungs of these animals, as measured by ELISA. Therefore, these data suggest that the production of IL-17 in MK-886-treated, immunized mice could contribute to the generation of a protective immune response after infection with M. tuberculosis.

Protection of chicken against very virulent IBDV provided by in ovo priming with DNA vaccine and boosting with killed vaccine and the adjuvant effects of plasmid-encoded chicken interleukin-2 and interferon-gamma

The aim of this study was to examine the efficacy of in ovo prime-boost vaccination against infectious bursal disease virus (IBDV) using a DNA vaccine to prime in ovo followed by a killed-vaccine boost post hatching. In addition, the adjuvant effects of plasmid-encoded chicken interleukin-2 and chicken interferon-gamma were tested in conjunction with the vaccine. A plasmid DNA vaccine (pcDNA-VP243) encoding the VP2, VP4, and VP3 proteins of the very virulent IBDV (vvIBDV) SH/92 strain was injected into the amniotic sac alone or in combination with a plasmid encoding chicken IL-2 (ChIL-2) or chicken IFN-gamma (ChIFN-gamma) at embryonation day 18, followed by an intramuscular injection of a commercial killed IBD vaccine at 1 week of age. The chickens were orally challenged with the vvIBDV SH/92 strain at 3 weeks of age and observed for 10 days. In ovo DNA immunization followed by a killed-vaccine boost provided significantly better immunity than the other options. No mortality was observed in this group after a challenge with the vvIBDV. The prime-boost strategy was moderately effective against bursal damage, which was measured by the bursa weight/body weight ratio, the presence of IBDV RNA, and the bursal lesion score. In ovo DNA vaccination with no boost did not provide sufficient immunity, and the addition of ChIL-2 or ChIFN-gamma did not enhance protective immunity. In the ConA-induced lymphocyte proliferation assay of peripheral blood lymphocyte collected 10 days post-challenge, there was greater proliferation responses in the DNA vaccine plus boost and DNA vaccine with ChIL-2 plus boost groups compared to the other groups. These findings suggest that priming with DNA vaccine and boosting with killed vaccine is an effective strategy for protecting chickens against vvIBDV.

The immunological effect of Ad/MDC-VP1 combined with DNA vaccine against Coxsackievirus infection / 中华微生物学和免疫学杂志

Objective To construct recombinant adenovirus Ad/MDC-VP1 and investigate its im-muno-boosting effect of the mice primed with the experimental DNA vaccine against Coxsackievirus infection. Methods The recombinant adenovirus Ad/MDC-VP1 was constructed and packaged. The Western blot analysis was used to verify the target protein. BALB/c mice were divided into four groups: Ad/MDC-VP1 group, pcDNA3/MDC-VP1 group, pcDNA3/MDC-VP1 prime-Ad/MDC-VP1 boost group and PBS group. The mice in each group were immunized intramuscularly. The titers of serum IgG and neutralizing antibody were tested by ELISA and trace neutralization assay, respectively. The lymphocytes proliferation activity and specific CTL cytotoxic activity were tested by CCK-8 assay. The mice in each group were challenged with le-thal dose of Coxsackievirus, and the assay of the serum virus titers and the observation of protection efficacy against Coxsackievirus infection were carried out. Results The recombinant adenovirus Ad/MDC-VP1 was successfully constructed and the target protein was expressed. It was observed that the titers of CVB3 VP1 specific antibody, lymphocyte stimulation index, CTL cytotoxicity activities and protection rate of the pcDNA3/MDC-VP1 prime-Ad/MDC-VP1 boost group were much higher than those of the rest groups( P < 0.05), and the titer of serum virus was lower after CVB3 challenged ( P < 0.05 ). Conclusion Both the cellular and humoral immune responses in mice could been significantly enhanced by the pcDNA3/MDC-VP1 prime-Ad/MDC-VP1 boost strategy.

The effects of immunity induced by BCG priming and tuberculosis fusion protein AMM vaccine boosting / 中华微生物学和免疫学杂志

Objective To investigate the boosting efficiency of a subunit vaccine consisting of the fusion protein Ag85B-Mpt64190-198-Mth8.4 (AMM) , dimethyl-dioctyldecyl ammonium bromide (DDA) and BCG polysaceharide nucleic acid (BCG-PSN) on the primed inoculation with BCG. Methods The AMM subunit vaccine was composed of fusion protein AMM, adjuvant DDA and BCG-PSN. The first mouse experi-mental group was immunized with BCG first, then boosted with the AMM subanit vaccine in the 10th week. The second experimental group was boosted with the AMM subunit vaccine in the 8th week and the 10th week respectively with a two weeks interval after the primed with BCG. Two control groups were treated re-spectively with physiological saline alone and BCG alone. After the primed inoculation, ELISPOT and ELISA were used for the detection of the cell-mediated and humoral immune response in week 14 and week 22 re-spectively. Furthermore, the immunized mice were challenged with live BCG to mimic tuberculosis infection in the 22nd week after the primed inoculation. Subsequently the T cell typing and humoral response were de-tected by flow cytometry and ELISA, respectively. Results ( 1 ) The level of secreting IFN-γ: 14 weeks af-ter the primed inoculation,with the stimulation of the specific antigen-Ag85B, the number of cells secreting IFN-γ in the second experimental group (135±14) was more than BCG alone immunized group (19±16), t = 10. 98, P < 0.01. In the 22nd week, the number of cells secreting IFN-γ in the second experimental group (208±11) was still more than BCG alone group (57±18), t =6.43, P <0.01. (2) The level of humoral immune response: the IgG1 antibody titer in the second experimental group was obviously higher than that in the first experimental group. However, the ratio of IgG2a to IgG1, as the index reflecting the Thl-type immune response, in the experimental group 2 was lower than that in the experimental group 1. (3) The contents of CD4+ CD25+ T cells after challenged with live BCG strain: the first and the second ex-perimental groups were both higher than the BCG alone group (t1 = 3.08, t2 = 3.16, P < 0.05 ). Conclu-sion Boosting the BCG-pfimed mice with tuberculosis AMM subunit vaccine twice can induce higher level of cell-mediated and humoral immune response than BCG alone, which could activate the regulative immune response at the same time.

In vivo CTL Activity Induced by Prime-boost Vaccination using Recombinant Vaccinia Virus and DNA Vaccine Expressing Epitope Specific for CD8+ T Cells

DNA vaccine approaches have been applied to generate the protective immunity against various pathogens. However, the strength of immune responses induced by DNA vaccine is weak compared with conventional vaccines. The primeboost vaccination using DNA vaccine and other viral vector has been suggested as one way to circumvent this limitation. In the present study, we used in vivo CTL activity assay to determine CD8+ T cell-mediated immunity induced by prime-boost vaccination with a DNA vaccine (gB498-505 DNA) and recombinant vaccinia virus (VVgB498-505) expressing gB498-505 epitope peptide (SSIEFARL) of herpes simplex virus type 1 (HSV-1) glycoprotein B (gB). The most potent in vivo CTL activity was induced in mice received VVgB498-505 when both gB498-505 and VVgB498-505 were used at priming step and boosted with the alternative vaccine vector expressing whole antigen protein (gBw). Priming with vaccine vector expressing gBw followed by the use of VVgB498-505 at boosting step also induced strong in vivo CTL activity. We also examined in vivo CTL activity after immunization of mice with epitope-expressing vaccine vector at both priming and boosting step. Curiously, in vivo CTL activity mediated by CD8+ T cells was strongly elicited at memory stage when animals were primed with VVgB498-505 and subsequently boosted with gB498-505 DNA. Because the use of VVgB498-505 at priming followed by boosting with gB498-505 DNA induced most optimal immunity, these results suggest that the order of vaccine type should be carefully considered when used vaccine type expressing only epitope for prime-boost vaccination.

Progress in Vaccine Development of Hepatitis C Virus / 中国生物工程杂志

Hepatitis C virus (HCV) accounts for the majority of cases of transfusion acquired hepatitis and may cause chronic hepatitis, cirrhosis and hepatocellular carcinoma. Currently, there is no vaccine against HCV and treatment is expensive and not always effective.The adaptive host immune response in viral clearance of HCV infection was described and the recent progress in vaccine development of HCV, focusing on the fields of DNA vaccine candidates, recombinant viral vectored vaccine candidates and combined (prime-boost) vaccine candidates were summarized. Some challenges and limitations of developing a HCV vaccine were also analysed. In summary, a promising approach of developing an experimental HCV vaccine to induce extremely potent and broad T-cell responses based on prime-boost strategy was presented.

CD8+ T Cell-mediated Immunity Induced by Heterologous Prime-boost Vaccination Based on DNA Vaccine and Recombinant Vaccinia Virus Expressing Epitope

BACKGROUND: DNA vaccination represents an anticipated approach for the control of numerous infectious diseases. Used alone, however, DNA vaccine is weak immunogen inferior to viral vectors. In recent, heterologous prime-boost vaccination leads DNA vaccines to practical reality. METHODS: We assessed prime-boost immunization strategies with a DNA vaccine (minigene, gB498-505 DNA) and recombinant vaccinia virus (vvgB498- 505) expressing epitope gB498-505 (SSIEFARL) of CD8+ T cells specific for glycoprotein B (gB) of herpes simplex virus (HSV). Animals were immunized primarily with gB498-505 epitope-expressing DNA vaccine/recombinant vaccinia virus and boosted with alternative vaccine type expressing entire Ag. RESULTS: In prime-boost protocols using vvgBw (recombinant vaccinia virus expressing entire Ag) and vvgB498-505, CD8+ T cell-mediated immunity was induced maximally at both acute and memory stages if primed with vvgBw and boosted with vvgB498-505 as evaluated by CTL activity, intracellular IFN-staining, and MHC class I tetramer staining. Similarly gB498-505 DNA prime-gBw DNA (DNA vaccine expressing entire Ag) boost immunization elicited the strongest CD8+ T cell responses in protocols based on DNA vaccine. However, the level of CD8+ T cell-mediated immunity induced with prime-boost vaccination using DNA vaccine expressing epitope or entire Ag was inferior to those based on vvgBw and vvgB498-505. Of particular interest CD8+ T cell-mediated immunity was optimally induced when vvgB498-505 was used to prime and gB DNA was used as alternative boost. Especially CD8+ T cell responses induced by such protocol was longer lasted than other protocols. CONCLUSION: These facts direct to search for the effective strategy to induce optimal CD8+ T cell-mediated immunity against cancer and viral infection.

Influence of Immunity Induced at Priming Step on Mucosal Immunization of Heterologous Prime-Boost Regimens

BACKGROUND: The usefulness of DNA vaccine at priming step of heterologous prime-boost vaccination led to DNA vaccine closer to practical reality. DNA vaccine priming followed by recombinant viral vector boosting via systemic route induces optimal systemic immunity but no mucosal immunity. Mucosal vaccination of the reversed protocol (recombinant viral vector priming-DNA vaccine boosting), however, can induce both maximal mucosal and systemic immunity. Here, we tried to address the reason why the mucosal protocol of prime-boost vaccination differs from that of systemic vaccination. METHODS: To address the importance of primary immunity induced at priming step, mice were primed with different doses of DNA vaccine or coadministration of DNA vaccine plus mucosal adjuvant, and immunity including serum IgG and mucosal IgA was then determined following boosting with recombinant viral vector. Next, to assess influence of humoral pre-existing immunity on boosting CD8+ T cell-mediated immunity, CD8+ T cell-mediated immunity in B cell-deficient (microK/O) mice immunized with prime-boost regimens was evaluated by CTL assay and IFN-gamma-producing cells. RESULTS: Immunity primed with recombinant viral vector was effectively boosted with DNA vaccine even 60 days later. In particular, animals primed by increasing doses of DNA vaccine or incorporating an adjuvant at priming step and boosted by recombinant viral vector elicited comparable responses to recombinant viral vector primed-DNA vaccine boosted group. Humoral pre-existing immunity was also unlikely to interfere the boosting effect of CD8+ T cell-mediated immunity by recombinant viral vector. CONCLUSION: This report provides the important point that optimally primed responses should be considered in mucosal immunization of heterologous prime-boost regimens for inducing the effective boosting at both mucosal and systemic sites.

Immune efficacy of Ag85B DNA/H37Ra prime-boost vaccination regimen for tuberculosis in mice / 重庆医科大学学报

Objective:To investigate the immune efficacy after sequential immunization with Mycobacterium tuberculosis DNA vaccine encoding mature form of Ag85B(pTB30m)and Mycobacterium tuberculosis H37Ra in mice.Methods:Much of highly pure plasmid DNA(pTB30m)extracted by alkaline lysis method was confirmed by restriction endonuclease digestion.Then,its DNA concentration and purity were determined by UV spectrophotometry.At various intervals(4weeks,8weeks)after sequential immunization,ELISA was used to detect the level of the serum antibody against PPD.Also,the spleen lymphocytes of mice were cultured with PPD in vitro.Lymphocyte transformation was detected by MTT assay.Results:Prepared pTB30m was highly pure and came to the needed concentration.Compared with Group Naive control,the specific antibody levels against PPD and the stimulation index(SI)of spleen lymphocytes were all statistically higher in Group DNA-85B/H37Ra(P0.05),as compared with Group DNA-85B/BCG,Group H37Ra and Group BCG,However,compared with Group H37Ra and Group BCG,the SI of mice was significantly larger in Group DNA-85B/H37Ra(P