Replication-defective Recombinant Adenovirus Research Articles

Rapid generation of a mouse model for evaluating on-target normal tissue toxicity of human CAR-T cells using replication-defective recombinant adenovirus

IntroductionThe on-target off-tumor toxicity of chimeric antigen receptor-engineered T cells (CAR-T) might lead to fatal side effects in cancer patients, which remains as a major obstacle to the clinical application of CAR-T immunotherapy. The off-tumor on-target normal tissue toxicity of CAR-T cells needs to be evaluated in preclinical studies using rational animal models. ObjectivesWe aim to develop a rational animal model for assessing the off-tumor on-target normal tissue toxicity of various CAR-T cell designs quickly. MethodsWe used a recombinant adenovirus type 5 carrying human HER2/ERBB2 (Ad5-HER2) or CD47 gene (Ad5-CD47) to rapidly generate a mouse model with tunable human antigen expression on normal liver tissue to determine immunotoxicity of traditional CAR-T and hypoxia-response CAR-T cells in vivo. ResultsThe obvious liver damage and lymphocyte infiltration were not observed in mice with human antigen-high livers 8 days post-infection. Interestingly, the lethal liver damage, systemic cytokine release and CAR-T cells infiltration in liver were only observed in mice that received traditional CAR-T cells, but not in hypoxia-response CAR-T cells. ConclusionAdenovirus-based expression of target antigen in normal mouse tissue may be a useful method for assessing on-target CAR-T cell toxicity in normal tissues, especially various CAR-T cell designs that have the potency of conditional regulation in tumor microenvironment (TME).

Boosting of SARS-CoV-2 immunity in nonhuman primates using an oral rhabdoviral vaccine

An orally active vaccine capable of boosting SARS-CoV-2 immune responses in previously infected or vaccinated individuals would help efforts to achieve and sustain herd immunity. Unlike mRNA-loaded lipid nanoparticles and recombinant replication-defective adenoviruses, replicating vesicular stomatitis viruses with SARS-CoV-2 spike glycoproteins (VSV-SARS2) were poorly immunogenic after intramuscular administration in clinical trials. Here, by G protein trans-complementation, we generated VSV-SARS2(+G) virions with expanded target cell tropism. Compared to parental VSV-SARS2, G-supplemented viruses were orally active in virus-naive and vaccine-primed cynomolgus macaques, powerfully boosting SARS-CoV-2 neutralizing antibody titers. Clinical testing of this oral VSV-SARS2(+G) vaccine is planned.

Vaccination With Recombinant Adenoviruses Expressing the Bluetongue Virus Subunits VP7 and VP2 Provides Protection Against Heterologous Virus Challenge.

Bluetongue virus (BTV) is the causative agent of a disease that affects domestic and wild ruminants and leads to critical economic losses. BTV is an arbovirus from the Reoviridae family that is typically transmitted by the bite of infected Culicoides midges. BTV possesses multiple serotypes (up to 28 have been described), and immunity to one serotype offers little cross-protection to other serotypes. The design of vaccines that provide protection across multiple serotypes is therefore highly desirable to control this disease. We previously reported that a recombinant replication-defective human adenovirus serotype 5 (Ad5) that expresses the VP7 inner core protein of BTV serotype 8 (Ad5VP7-8) induced T-cell responses and provided protection. In the present work, we evaluated as BTV vaccine the combination of Ad5VP7-8 with another recombinant Ad5 that expresses the outer core protein VP2 from BTV-1 (Ad5VP2-1). The combination of Ad5VP2-1 and Ad5VP7-8 protected against homologous BTV challenge (BTV-1 and BTV-8) and partially against heterologous BTV-4 in a murine model. Cross-reactive anti-BTV immunoglobulin G (IgG) were detected in immunized animals, but no significant titers of neutralizing antibodies were elicited. The Ad5VP7-8 immunization induced T-cell responses that recognized all three serotypes tested in this study and primed cytotoxic T lymphocytes specific for VP7. This study further confirms that targeting antigenic determinant shared by several BTV serotypes using cellular immunity could help develop multiserotype BTV vaccines.

Boosting BCG with proteins or rAd5 does not enhance protection against tuberculosis in rhesus macaques

Tuberculosis (TB) is the leading cause of death from infection worldwide. The only approved vaccine, BCG, has variable protective efficacy against pulmonary TB, the transmissible form of the disease. Therefore, improving this efficacy is an urgent priority. This study assessed whether heterologous prime-boost vaccine regimens in which BCG priming is boosted with either (i) protein and adjuvant (M72 plus AS01E or H56 plus CAF01) delivered intramuscularly (IM), or (ii) replication-defective recombinant adenovirus serotype 5 (Ad5) expressing various Mycobacterium tuberculosis (Mtb) antigens (Ad5(TB): M72, ESAT-6/Ag85b, or ESAT-6/Rv1733/Rv2626/RpfD) administered simultaneously by IM and aerosol (AE) routes, could enhance blood- and lung-localized T-cell immunity and improve protection in a nonhuman primate (NHP) model of TB infection. Ad5(TB) vaccines administered by AE/IM routes following BCG priming elicited ~10–30% antigen-specific CD4 and CD8 T-cell multifunctional cytokine responses in bronchoalveolar lavage (BAL) but did not provide additional protection compared to BCG alone. Moreover, AE administration of an Ad5(empty) control vector after BCG priming appeared to diminish protection induced by BCG. Boosting BCG by IM immunization of M72/AS01E or H56:CAF01 elicited ~0.1–0.3% antigen-specific CD4 cytokine responses in blood with only a transient increase of ~0.5–1% in BAL; these vaccine regimens also failed to enhance BCG-induced protection. Taken together, this study shows that boosting BCG with protein/adjuvant or Ad-based vaccines using these antigens, by IM or IM/AE routes, respectively, do not enhance protection against primary infection compared with BCG alone, in the highly susceptible rhesus macaque model of tuberculosis.

Evaluation of recombinant adenovirus vaccines based on glycoprotein D and truncated UL25 against herpes simplex virus type 2 in mice.

The high prevalence of herpes simplex virus 2 (HSV-2) infections in humans necessitates the development of a safe and effective vaccine that will need to induce vigorous T-cell responses to control viral infection and transmission. We designed rAd-gD2, rAd-gD2ΔUL25, and rAd-ΔUL25 to investigate whether recombinant replication-defective adenoviruses vaccine could induce specific T-cell responses and protect mice against intravaginal HSV-2 challenge compared with FI-HSV-2. In the present study, recombinant adenovirus-based HSV-2 showed higher reductions in mortality and stronger antigen-specific T-cell responses compared with FI-HSV-2 and the severity of genital lesions in mice immunized with rAd-gD2ΔUL25 was significantly decreased by eliciting IFN-γ-secreting T-cell responses compared with rAd-gD2 and rAd-ΔUL25 groups. Our results demonstrated the immunogenicity and protective efficacy of recombinant adenovirus vaccines in acute HSV-2 infection following intravaginal challenge in mice.

Chimpanzee Adenovirus Vector Ebola Vaccine

BackgroundThe unprecedented 2014 epidemic of Ebola virus disease (EVD) prompted an international response to accelerate the availability of a preventive vaccine. A replication-defective recombinant chimpanzee adenovirus type 3–vectored ebolavirus vaccine (cAd3-EBO), encoding the glycoprotein from Zaire and Sudan species, that offers protection in the nonhuman primate model, was rapidly advanced into phase 1 clinical evaluation.MethodsWe conducted a phase 1, dose-escalation, open-label trial of cAd3-EBO. Twenty healthy adults, in sequentially enrolled groups of 10 each, received vaccination intramuscularly in doses of 2×1010 particle units or 2×1011 particle units. Primary and secondary end points related to safety and immunogenicity were assessed throughout the first 8 weeks after vaccination; in addition, longer-term vaccine durability was assessed at 48 weeks after vaccination.ResultsIn this small study, no safety concerns were identified; however, transient fever developed within 1 day after vaccination in two participants who had received the 2×1011 particle-unit dose. Glycoprotein-specific antibodies were induced in all 20 participants; the titers were of greater magnitude in the group that received the 2×1011 particle-unit dose than in the group that received the 2×1010 particle-unit dose (geometric mean titer against the Zaire antigen at week 4, 2037 vs. 331; P=0.001). Glycoprotein-specific T-cell responses were more frequent among those who received the 2×1011 particle-unit dose than among those who received the 2×1010 particle-unit dose, with a CD4 response in 10 of 10 participants versus 3 of 10 participants (P=0.004) and a CD8 response in 7 of 10 participants versus 2 of 10 participants (P=0.07) at week 4. Assessment of the durability of the antibody response showed that titers remained high at week 48, with the highest titers in those who received the 2×1011 particle-unit dose.ConclusionsReactogenicity and immune responses to cAd3-EBO vaccine were dose-dependent. At the 2×1011 particle-unit dose, glycoprotein Zaire–specific antibody responses were in the range reported to be associated with vaccine-induced protective immunity in challenge studies involving nonhuman primates, and responses were sustained to week 48. Phase 2 studies and efficacy trials assessing cAd3-EBO are in progress. (Funded by the Intramural Research Program of the National Institutes of Health; VRC 207 ClinicalTrials.gov number, NCT02231866.)

Antitumor Efficacy of SLPI Promoter-Controlled Expression of Artificial microRNA Targeting EGFR in a Squamous Cell Carcinoma Cell Line.

The purpose of this study was to develop a recombinant adenovirus with secretory leukoprotease inhibitor (SLPI) promoter-controlled expression for gene therapy of squamous cell carcinoma (SCC). An artificial microRNA targeting epidermal growth factor receptor (EGFR) was designed, and used to construct a replication-defective recombinant adenovirus with SLPI promoter-controlled expression. The silencing efficiency of this vector (Ad-SLPI-EGFRamiR) was detected in Hep-2 cells. Western blotting showed that the expression of 170 kD EGFR was significantly reduced in Hep-2 cells 72h after infection with Ad-SLPI-EGFRamiR. At a multiplicity of infection (MOI) of 200 pfu/cell, proliferation of Hep-2 cells was highly inhibited by Ad-SLPI-EGFRamiR (inhibition rate: ~70%). The apoptosis rate of Hep-2 cells at 72h after infection with Ad-SLPI-EGFRamiR at a MOI 35 pfu/cell was 32.8%. The adenovirus constructed was able to specifically inhibit the growth of SCC cells in vitro.

Protective Efficacy in Sheep of Adenovirus-Vectored Vaccines against Bluetongue Virus Is Associated with Specific T Cell Responses.

Bluetongue virus (BTV) is an economically important Orbivirus of the Reoviridae family that causes a hemorrhagic disease in ruminants. Its control has been achieved by inactivated-vaccines that have proven to protect against homologous BTV challenge although unable to induce long-term immunity. Therefore, a more efficient control strategy needs to be developed. Recombinant adenovirus vectors are lead vaccine candidates for protection of several diseases, mainly because of their potency to induce potent T cell immunity. Here we report the induction of humoral and T-cell mediated responses able to protect animals against BTV challenge by recombinant replication-defective human adenovirus serotype 5 (Ad5) expressing either VP7, VP2 or NS3 BTV proteins. First we used the IFNAR(-/-) mouse model system to establish a proof of principle, and afterwards we assayed the protective efficacy in sheep, the natural host of BTV. Mice were completely protected against BTV challenge, developing humoral and BTV-specific CD8+- and CD4+-T cell responses by vaccination with the different rAd5. Sheep vaccinated with Ad5-BTV-VP2 and Ad5-BTV-VP7 or only with Ad5-BTV-VP7 and challenged with BTV showed mild disease symptoms and reduced viremia. This partial protection was achieved in the absence of neutralizing antibodies but strong BTV-specific CD8+ T cell responses in those sheep vaccinated with Ad5-BTV-VP7. These data indicate that rAd5 is a suitable vaccine vector to induce T cell immunity during BTV vaccination and provide new data regarding the relevance of T cell responses in protection during BTV infection.

Co-expression of the C-terminal domain of Yersinia enterocolitica invasin enhances the efficacy of classical swine-fever-vectored vaccine based on human adenovirus.

The use of adenovirus vector-based vaccines is a promising approach for generating antigen-specific immune responses. Improving vaccine potency is necessary in other approaches to address their inadequate protection for the majority of infectious diseases. This study is the first to reconstruct a recombinant replication-defective human adenovirus co-expressing E2 and invasin C-terminal (InvC) glycoproteins (rAd-E2-InvC). rAd-E2-InvC with 2 x 10(6) TCID50 was intramuscularly administered two times to CSFV-free pigs at 14 day intervals. No adverse clinical reactions were observed in any of the pigs after the vaccination. The CSFV E2-specific antibody titer was significantly higher in the rAd-E2-InvC group than that in the rAdV-E2 group as measured by NPLA and blocking ELISA. Pigs immunized with rAd-E2-InvC were completely protected against lethal challenge. Neither CSFV RNA nor pathological changes were detected in the tissues after CSFV challenge. These results demonstrate that rAd-E2-InvC could be an alternative to the existing CSF vaccine. Moreover, InvC that acts as an adjuvant could enhance the immunogenicity of rAdV-E2 and induce high CSFV E2-specific antibody titer and protection level.

Aerosol vaccination with AERAS-402 elicits robust cellular immune responses in the lungs of rhesus macaques but fails to protect against high-dose Mycobacterium tuberculosis challenge.

Development of a vaccine against pulmonary tuberculosis may require immunization strategies that induce a high frequency of Ag-specific CD4 and CD8 T cells in the lung. The nonhuman primate model is essential for testing such approaches because it has predictive value for how vaccines elicit responses in humans. In this study, we used an aerosol vaccination strategy to administer AERAS-402, a replication-defective recombinant adenovirus (rAd) type 35 expressing Mycobacterium tuberculosis Ags Ag85A, Ag85B, and TB10.4, in bacillus Calmette-Guérin (BCG)-primed or unprimed rhesus macaques. Immunization with BCG generated low purified protein derivative-specific CD4 T cell responses in blood and bronchoalveolar lavage. In contrast, aerosolized AERAS-402 alone or following BCG induced potent and stable Ag85A/b-specific CD4 and CD8 effector T cells in bronchoalveolar lavage that largely produced IFN-γ, as well as TNF and IL-2. Such responses induced by BCG, AERAS-402, or both failed to confer overall protection following challenge with 275 CFUs M. tuberculosis Erdman, although vaccine-induced responses associated with reduced pathology were observed in some animals. Anamnestic T cell responses to Ag85A/b were not detected in blood of immunized animals after challenge. Overall, our data suggest that a high M. tuberculosis challenge dose may be a critical factor in limiting vaccine efficacy in this model. However, the ability of aerosol rAd immunization to generate potent cellular immunity in the lung suggests that using different or more immunogens, alternative rAd serotypes with enhanced immunogenicity, and a physiological challenge dose may achieve protection against M. tuberculosis.

Vaccination with recombinant adenoviruses expressing the peste des petits ruminants virus F or H proteins overcomes viral immunosuppression and induces protective immunity against PPRV challenge in sheep.

Peste des petits ruminants (PPR) is a highly contagious disease of small ruminants caused by the Morbillivirus peste des petits ruminants virus (PPRV). Two recombinant replication-defective human adenoviruses serotype 5 (Ad5) expressing either the highly immunogenic fusion protein (F) or hemagglutinin protein (H) from PPRV were used to vaccinate sheep by intramuscular inoculation. Both recombinant adenovirus vaccines elicited PPRV-specific B- and T-cell responses. Thus, neutralizing antibodies were detected in sera from immunized sheep. In addition, we detected a significant antigen specific T-cell response in vaccinated sheep against two different PPRV strains, indicating that the vaccine induced heterologous T cell responses. Importantly, no clinical signs and undetectable virus shedding were observed after virulent PPRV challenge in vaccinated sheep. These vaccines also overcame the T cell immunosuppression induced by PPRV in control animals. The results indicate that these adenovirus constructs could be a promising alternative to current vaccine strategies for the development of PPRV DIVA vaccines.

Hepatitis C virus-induced hepatocyte cell death and protection by inhibition of apoptosis.

Chronic hepatitis C virus (HCV) infection results in progressive liver fibrosis leading to cirrhosis and liver cancer. The mechanism for this remains unclear but hepatocyte apoptosis is thought to play a major role. Hepatocyte apoptosis in human liver tissue was determined by immunohistochemistry for cytokeratin 18 (M30 CytoDEATH) and cleaved poly(ADP-ribose) polymerase (PARP). In vitro studies were performed with replication-defective recombinant adenoviruses expressing HCV proteins (rAdHCV) to study the effects of HCV on cell death in Huh7 cells, primary mouse hepatocytes (PMoHs) and primary human hepatocytes (PHHs). Cell viability and apoptosis were studied using crystal violet assays and Western blots probed for cleaved caspase-3 and cleaved PARP, with and without treatment with the pan-caspase inhibitor Q-VD-OPh and necrostatin-1. Liver tissue of HCV-infected patients expressed elevated levels of apoptotic markers compared with HCV-negative patients. rAdHCV infection reduced cell viability compared with uninfected controls and cells infected with control virus (rAdGFP). Huh7, PMoHs and PHHs infected with rAdHCV showed significantly increased levels of apoptotic markers compared with uninfected controls and rAdGFP-infected cells. In rAdHCV-infected Huh7, treatment with Q-VD-OPh and necrostatin-1 both improved cell viability. Q-VD-Oph also reduced cleaved PARP in rAdHCV-infected Huh7 and PMoHs. Hepatocyte apoptosis is known to be increased in the livers of HCV-infected patients. HCV promoted cell death in primary and immortalized hepatocytes, and this was inhibited by Q-VD-OPh and necrostatin-1. These findings indicate that HCV-induced cell death occurs by both apoptosis and necroptosis, and provide new insights into the mechanisms of HCV-induced liver injury.

S1 of distinct IBV population expressed from recombinant adenovirus confers protection against challenge.

Protective properties of three distinct infectious bronchitis virus (IBV) Ark Delmarva poultry industry (ArkDPI) S1 proteins encoded from replication-defective recombinant adenovirus vectors were investigated. Using a suboptimal dose of each recombinant virus, we demonstrated that IBV S1 amino acid sequences showing > or = 95.8% amino acid identity to the S1 of the challenge strain differed in their ability at conferring protection. Indeed, the S1 sequence of the IBV population previously designated C4 (AdIBVS1.C4), which protected the most poorly, differs from the S1 sequence of population C2 (AdIBVS1.C2), which provided the highest protection, only at amino acid position 56. The fact that a change in one amino acid in this region significantly altered the induction of a protective immune response against this protein provides evidence that the first portion of S1 displays relevant immunoprotective epitopes. Use of an optimal dose of AdIBVS1.C2 effectively protected chickens from clinical signs and significantly reduced viral load after IBV Ark virulent challenge. Moreover, increased numbers of both IgA and IgG IBV-specific antibody secreting lymphocytes were detected in the spleen after challenge. The increased response detected for both IgA and IgG lymphocytes after challenge might be explained by vaccine-induced B memory cells. The fact that a single vaccination with Ad/IBVS1.C2 provides protection against IBV challenge is promising, because Ad-vectored vaccines can be mass delivered by in ovo inoculation using automated in ovo injectors.

Enhancement of nasal HIV vaccination with adenoviral vector-based nanocomplexes using mucoadhesive and DC-targeting adjuvants.

To investigate the vaccine effect of a replication-defective recombinant adenovirus 5 (rAd5)-based nanocomplex with chitooligosaccharides (Oligo) and mannosylated polyethyleneimine-triethyleneglycol (mPEI) as adjuvants for human immunodeficiency virus (HIV) infection. Physical characteristics were determined through detecting the size, zeta potential and morphology of Oligo-mPEI-rAd5 nanocomplex, and in vitro vaccine uptake and transduction efficiency were estimated. Nanocomplexes were then administered intranasally to Balb/c mice to evaluate in vivo rAd5 residence in nasal cavity and HIVgag-specific immune responses using cytotoxic T lymphocyte (CTL), intracellular cytokine staining (ICS) and ELISA assay. The mucoadhesivity of Oligo prolonged nasal residence time, while the dendritic cell (DC) specificity of mPEI improved vaccine uptake. These two adjuvants jointly enhanced transduction efficiency of rAd5. Oligo-mPEI-rAd5 nanocomplex elicited potent HIVgag-specific CTL response and increased IFN-γ positive CD8(+)T and IL-4 positive CD4(+)T cells, indicating high cellular immune responses. This vaccine candidate also led to strong humoral immune responses (IgG/IgG1/IgG2a) with balanced Th1/Th2 CD4(+)T cell activity. Moreover, mice nasally immunized with Oligo-mPEI-rAd5 showed higher levels of SIgA in nasal washes than did mice immunized with rAd5. Intranasal delivery of Oligo-mPEI-rAd5 with a prime-boost regimen is a potential immunization for HIV infection, inducing HIVgag-specific cellular, humoral and mucosal immune responses.

Cytotoxic T lymphocytes elicited by dendritic cell-targeted delivery of human papillomavirus type-16 E6/E7 fusion gene exert lethal effects on CaSki cells.

Human papillomavirus (HPV) is the primary etiologic agent of cervical cancer. Consideration of safety and non human leukocyte antigen restriction, protein vaccine has become the most likely form of HPV therapeutic vaccine, although none have so far been reported as effective. Since tumor cells consistently express the two proteins E6 and E7, most therapeutic vaccines target one or both of them. In this study, we fabricated DC vaccines by transducing replication-defective recombinant adenoviruses expressing E6/E7 fusion gene of HPV-16, to investigate the lethal effects of specific cytotoxic T lymphocytes (CTL) against CaSki cells in vitro. Mouse immature dendritic cells (DC) were generated from bone marrow, and transfected with pAd-E6/E7 to prepare a DC vaccine and to induce specific CTL. The surface expression of CD40, CD68, MHC II and CD11c was assessed by flow cytometry (FCM), and the lethal effects of CTL against CaSki cells were determined by DAPI, FCM and CCK-8 methods. Immature mouse DC was successfully transfected by pAd-E6/E7 in vitro, and the transfecting efficiency was 40%-50%. A DC vaccine was successfully prepared and was used to induce specific CTL. Experimental results showed that the percentage of apoptosis and killing rate of CaSki cells were significantly increased by coculturing with the specific CTL (p <0.05). These results illustrated that a DC vaccine modified by HPV-16 E6/E7 gene can induce apoptosis of CaSki cells by inducing CTL, which may be used as a new strategy for biological treatment of cervical cancer.

Recombinant adenovirus expressing the haemagglutinin of peste des petits ruminants virus (PPRV) protects goats against challenge with pathogenic virus; a DIVA vaccine for PPR

Peste des petits ruminants virus (PPRV) is a morbillivirus that can cause severe disease in sheep and goats, characterised by pyrexia, pneumo-enteritis, and gastritis. The socio-economic burden of the disease is increasing in underdeveloped countries, with poor livestock keepers being affected the most. Current vaccines consist of cell-culture attenuated strains of PPRV, which induce a similar antibody profile to that induced by natural infection. Generation of a vaccine that enables differentiation of infected from vaccinated animals (DIVA) would benefit PPR control and eradication programmes, particularly in the later stages of an eradication campaign and for countries where the disease is not endemic. In order to create a vaccine that would enable infected animals to be distinguished from vaccinated ones (DIVA vaccine), we have evaluated the immunogenicity of recombinant fowlpox (FP) and replication-defective recombinant human adenovirus 5 (Ad), expressing PPRV F and H proteins, in goats. The Ad constructs induced higher levels of virus-specific and neutralising antibodies, and primed greater numbers of CD8+ T cells than the FP-vectored vaccines. Importantly, a single dose of Ad-H, with or without the addition of Ad expressing ovine granulocyte macrophage colony-stimulating factor and/or ovine interleukin-2, not only induced strong antibody and cell-mediated immunity but also completely protected goats against challenge with virulent PPRV, 4 months after vaccination. Replication-defective Ad-H therefore offers the possibility of an effective DIVA vaccine.

Enhancing mucosal immunity in mice by recombinant adenovirus expressing major epitopes of porcine circovirus-2 capsid protein delivered with cytosine-phosphate-guanosine oligodeoxynucleotides

A recombinant replication-defective adenovirus expressing the major epitopes of porcine circovirus-2 (PCV-2) capsid protein (rAd/Cap/518) was previously constructed and shown to induce mucosal immunity in mice following intranasal delivery. In the present study, immune responses induced by intranasal immunization with a combination of rAd/Cap/518 and cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) were evaluated in mice. The levels of PCV-2-specific IgG in serum and IgA in saliva, lung, and intestinal fluids were significantly higher in the group immunized with rAd/Cap/518 and CpG ODN than animals immunized with rAd/Cap/518 alone. The frequencies of IL-2-secreting CD4+ T cells and IFN-γ-producing CD8+ T cells were significantly higher in the combined immunization group than mice immunized with rAd/Cap/518 alone. The frequencies of CD3+, CD3+CD4+CD8-, and CD3+CD4-CD8+ T cells in the combined immunization group were similar to that treated with CpG ODN alone, but significantly higher than mice that did not receive CpG ODN. PCV-2 load after challenge in the combined immunization group was significantly lower than that in the phosphate-buffered saline placebo group and approximately 7-fold lower in the group treated with CpG ODN alone. These results indicate that rAd/Cap/518 combined with CpG ODN can enhance systemic and local mucosal immunity in mice, and represent a promising synergetic mucosal vaccine against PCV-2.

Immunogenicity of a Prime-Boost Vaccine Containing the Circumsporozoite Proteins of Plasmodium vivax in Rodents

Plasmodium vivax is the most widespread and the second most prevalent malaria-causing species in the world. Current measures used to control the transmission of this disease would benefit from the development of an efficacious vaccine. In the case of the deadly parasite P. falciparum, the recombinant RTS,S vaccine containing the circumsporozoite antigen (CSP) consistently protects 30 to 50% of human volunteers against infection and is undergoing phase III clinical trials in Africa with similar efficacy. These findings encouraged us to develop a P. vivax vaccine containing the three circulating allelic forms of P. vivax CSP. Toward this goal, we generated three recombinant bacterial proteins representing the CSP alleles, as well as a hybrid polypeptide called PvCSP-All-CSP-epitopes. This hybrid contains the conserved N and C termini of P. vivax CSP and the three variant repeat domains in tandem. We also generated simian and human recombinant replication-defective adenovirus vectors expressing PvCSP-All-CSP-epitopes. Mice immunized with the mixture of recombinant proteins in a formulation containing the adjuvant poly(I·C) developed high and long-lasting serum IgG titers comparable to those elicited by proteins emulsified in complete Freund's adjuvant. Antibody titers were similar in mice immunized with homologous (protein-protein) and heterologous (adenovirus-protein) vaccine regimens. The antibodies recognized the three allelic forms of CSP, reacted to the repeated and nonrepeated regions of CSP, and recognized sporozoites expressing the alleles VK210 and VK247. The vaccine formulations described in this work should be useful for the further development of an anti-P. vivax vaccine.

Two replication-defective adenoviral vaccine vectors for the induction of immune responses to PPRV

Peste des petits ruminants is a highly contagious disease of small ruminants caused by a Morbillivirus, peste des petits ruminants virus (PPRV). Two recombinant replication-defective human adenovirus serotype 5 (Ad5) containing the highly immunogenic fusion protein (F) and hemaglutinine protein (H) genes from PPRV were constructed. HEK293A cells infected with either virus (Ad5-PPRV-F or -H) express F and H proteins respectively. These viruses were used to vaccinate mice by intramuscular inoculation. Both viruses elicited PPRV-specific B- and T-cell responses. Thus, after two immunizations, sera from immunized mice elicited neutralizing antibody response, indicating that this approach has the potential to confer protective immunity. In addition, we detected a significant antigen specific CD4+ and CD8+ T-cell response in mice vaccinated with either virus. These results indicate that these adenovirus constructs offer a promising alternative to current vaccine strategies for the development of PPRV DIVA vaccines.

Development of a dendritic cell vaccine encoding multiple cytotoxic T lymphocyte epitopes targeting hepatitis C virus

The aim of the present study was to develop a dendritic cell (DC) vaccine encoding hepatitis C virus (HCV) multiple cytotoxic T lymphocyte (CTL) epitopes that can stimulate T cell responses in vitro, and can be used for immunization in vivo. DCs were infected with recombinant replication-defective adenoviruses (Ads) expressing 2 HCV sequences fused with green fluorescent protein (GFP) and FLAG tags. One sequence (sequence 1) contained the HCV CTL epitopes, NS4B 1793-1801 and P7 774-782, as well as the HCV Th epitope, NS3 1248-1261. A second sequence (sequence 2) was the positive epitope control which contained HCV core 35-44, core 132-140 and NS3 1248-1261. The efficiency of infection was detected by flow cytometry and the expression of HCV epitopes in the DCs was confirmed by RT-PCR and western blot analysis. Ad infection significantly enhanced DC maturation and interleukin (IL)-12p70 production, resulting in T cell proliferation and increased interferon-γ secretion. The CTLs stimulated by Ad-infected DCs specifically killed Huh7.5 human hepatoma cells. The recombinant Ad-expressing multiple CTL HCV epitopes effectively infected the DCs in vitro and promoted T cell antiviral immune responses, thereby laying the foundation for the development of anti-HCV DC vaccines.