Replication-defective Human Type Research Articles

Comparative evaluation of protective immunity against Francisella tularensis induced by subunit or adenovirus-vectored vaccines.

Tularemia is a highly contagious disease caused by infection with Francisella tularensis (Ft), a pathogenic intracellular gram-negative bacterium that infects a wide range of animals and causes severe disease and death in people, making it a public health concern. Vaccines are the most effective way to prevent tularemia. However, there are no Food and Drug Administration (FDA)-approved Ft vaccines thus far due to safety concerns. Herein, three membrane proteins of Ft, Tul4, OmpA, and FopA, and a molecular chaperone, DnaK, were identified as potential protective antigens using a multifactor protective antigen platform. Moreover, the recombinant DnaK, FopA, and Tul4 protein vaccines elicited a high level of IgG antibodies but did not protect against challenge. In contrast, protective immunity was elicited by a replication-defective human type 5 adenovirus (Ad5) encoding the Tul4, OmpA, FopA, and DnaK proteins (Ad5-Tul4, Ad5-OmpA, Ad5-FopA, and Ad5-DnaK) after a single immunization, and all Ad5-based vaccines stimulated a Th1-biased immune response. Moreover, intramuscular and intranasal vaccination with Ad5-Tul4 using the prime-boost strategy effectively eliminated Ft lung, spleen and liver colonization and provided nearly 80% protection against intranasal challenge with the Ft live vaccine strain (LVS). Only intramuscular, not intranasal vaccination, with Ad5-Tul4 protected mice from intraperitoneal challenge. This study provides a comprehensive comparison of protective immunity against Ft provided by subunit or adenovirus-vectored vaccines and suggests that mucosal vaccination with Ad5-Tul4 may yield desirable protective efficacy against mucosal infection, while intramuscular vaccination offers greater overall protection against intraperitoneal tularemia.

Treatment with Ad5-Porcine Interferon-α Attenuates Ebolavirus Disease in Pigs.

Under experimental conditions, pigs infected with Ebola Virus (EBOV) develop disease and can readily transmit the virus to non-human primates or pigs. In the event of accidental or intentional EBOV infection of domestic pigs, complex and time-consuming safe depopulation and carcass disposal are expected. Delaying or preventing transmission through a reduction in viral shedding is an absolute necessity to limit the spread of the virus. In this study, we tested whether porcine interferon-α or λ3 (porIFNα or porIFNλ3) delivered by a replication-defective human type 5 adenovirus vector (Ad5-porIFNα or Ad5-porIFNλ3) could limit EBOV replication and shedding in domestic pigs. Our results show that pigs pre-treated with Ad5-porIFNα did not develop measurable clinical signs, did not shed virus RNA, and displayed strongly reduced viral RNA load in tissues. A microarray analysis of peripheral blood mononuclear cells indicated that Ad5-porIFNα treatment led to clear upregulation in immune and inflammatory responses probably involved in protection against disease. Our results indicate that administration of Ad5-porIFNα can potentially be used to limit the spread of EBOV in pigs.

A single dose of an adenovirus-vectored vaccine provides protection against SARS-CoV-2 challenge

The unprecedented coronavirus disease 2019 (COVID-19) epidemic has created a worldwide public health emergency, and there is an urgent need to develop an effective vaccine to control this severe infectious disease. Here, we find that a single vaccination with a replication-defective human type 5 adenovirus encoding the SARS-CoV-2 spike protein (Ad5-nCoV) protect mice completely against mouse-adapted SARS-CoV-2 infection in the upper and lower respiratory tracts. Additionally, a single vaccination with Ad5-nCoV protects ferrets from wild-type SARS-CoV-2 infection in the upper respiratory tract. This study suggests that the mucosal vaccination may provide a desirable protective efficacy and this delivery mode is worth further investigation in human clinical trials.

A Replication-Defective Human Type 5 Adenovirus-Based Trivalent Vaccine Confers Complete Protection against Plague in Mice and Nonhuman Primates.

Currently, no plague vaccine exists in the United States for human use. The capsular antigen (Caf1 or F1) and two type 3 secretion system (T3SS) components, the low-calcium-response V antigen (LcrV) and the needle protein YscF, represent protective antigens of Yersinia pestis We used a replication-defective human type 5 adenovirus (Ad5) vector and constructed recombinant monovalent and trivalent vaccines (rAd5-LcrV and rAd5-YFV) that expressed either the codon-optimized lcrV or the fusion gene designated YFV (consisting of ycsF, caf1, and lcrV). Immunization of mice with the trivalent rAd5-YFV vaccine by either the intramuscular (i.m.) or the intranasal (i.n.) route provided protection superior to that with the monovalent rAd5-LcrV vaccine against bubonic and pneumonic plague when animals were challenged with Y. pestis CO92. Preexisting adenoviral immunity did not diminish the protective response, and the protection was always higher when mice were administered one i.n. dose of the trivalent vaccine (priming) followed by a single i.m. booster dose of the purified YFV antigen. Immunization of cynomolgus macaques with the trivalent rAd5-YFV vaccine by the prime-boost strategy provided 100% protection against a stringent aerosol challenge dose of CO92 to animals that had preexisting adenoviral immunity. The vaccinated and challenged macaques had no signs of disease, and the invading pathogen rapidly cleared with no histopathological lesions. This is the first report showing the efficacy of an adenovirus-vectored trivalent vaccine against pneumonic plague in mouse and nonhuman primate (NHP) models.

A human type 5 adenovirus-based Trypanosoma cruzi therapeutic vaccine re-programs immune response and reverses chronic cardiomyopathy.

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is a prototypical neglected tropical disease. Specific immunity promotes acute phase survival. Nevertheless, one-third of CD patients develop chronic chagasic cardiomyopathy (CCC) associated with parasite persistence and immunological unbalance. Currently, the therapeutic management of patients only mitigates CCC symptoms. Therefore, a vaccine arises as an alternative to stimulate protective immunity and thereby prevent, delay progression and even reverse CCC. We examined this hypothesis by vaccinating mice with replication-defective human Type 5 recombinant adenoviruses (rAd) carrying sequences of amastigote surface protein-2 (rAdASP2) and trans-sialidase (rAdTS) T. cruzi antigens. For prophylactic vaccination, naïve C57BL/6 mice were immunized with rAdASP2+rAdTS (rAdVax) using a homologous prime/boost protocol before challenge with the Colombian strain. For therapeutic vaccination, rAdVax administration was initiated at 120 days post-infection (dpi), when mice were afflicted by CCC. Mice were analyzed for electrical abnormalities, immune response and cardiac parasitism and tissue damage. Prophylactic immunization with rAdVax induced antibodies and H-2Kb-restricted cytotoxic and interferon (IFN)γ-producing CD8+ T-cells, reduced acute heart parasitism and electrical abnormalities in the chronic phase. Therapeutic vaccination increased survival and reduced electrical abnormalities after the prime (analysis at 160 dpi) and the boost (analysis at 180 and 230 dpi). Post-therapy mice exhibited less heart injury and electrical abnormalities compared with pre-therapy mice. rAdVax therapeutic vaccination preserved specific IFNγ-mediated immunity but reduced the response to polyclonal stimuli (anti-CD3 plus anti-CD28), CD107a+ CD8+ T-cell frequency and plasma nitric oxide (NO) levels. Moreover, therapeutic rAdVax reshaped immunity in the heart tissue as reduced the number of perforin+ cells, preserved the number of IFNγ+ cells, increased the expression of IFNγ mRNA but reduced inducible NO synthase mRNA. Vaccine-based immunostimulation with rAd might offer a rational alternative for re-programming the immune response to preserve and, moreover, recover tissue injury in Chagas’ heart disease.

P16INK4a expression mediated by recombinant adenovirus can induce senescence of A549 cells

To construct E1-deletion and replication-defective human type 5 recombinant adenovirus vector and to study the effect of p16INK4a on proliferation and aging of A549 cells. p16INK4a cDNA was cloned into pAdCMV to construct recombinant pAdCMV p16INK4a, which was co-transfected into 293 cell together with pJM17. The recombinant p16INK4a adenovirus (Ad-p16INK4a) was generated by homologous recombination and identified with duplex PCR. Lung cancer cell A549, which has a homozygous deletion of p16INK4a gene, was infected with the prepared Ad-p16INK4a virus. X-gal staining and TRAP-ELISA were used for detecting senescence-associated beta-galactosidase and telomerase activities in A549 cells. Immunohistochemical staining and Western blot indicated that p16INK4a gene was transferred into A549 cell with more than 95% efficiency by recombinant adenovirus and p16INK4a protein was expressed at a high level- p16INK4a could markedly inhibit growth of A549 cells, induced expression of senescence-associated beta-galactosidase and suppressed telomerase activity in A549 cells. Recombinant adenovirus vector could efficiently mediate transfer and expression of foreign genes in human cell and could be used for gene immunization and gene therapy; p16INK4a could inhibit A549 cell growth and induce its replicative senescence.

Adenoviral-expressed GP5 of porcine respiratory and reproductive syndrome virus differs in its cellular maturation from the authentic viral protein but maintains known biological functions.

Summary. The ORFs 5, 6 and 7, encoding for the three major structural proteins, GP5, M and N, of the IAF-Klop strain of PRRSV were cloned and expressed in 293 cells using replication-defective human type 5 adenoviral vectors (hAdVs). Although the M protein gene could be cloned into hAdVs and expressed constituvely in 293 cells under the control of the hCMV immediate early promotor/enhancer, hAdVs expressing N and GP5 proteins, which appeared to be toxic or interfered with adenovirus replication, could only be generated by inclusion of a tetracycline-regulatable promotor in the transfer vector pAdTR5. The recombinant (rec) proteins appeared similar to the authentic viral proteins in regards to their M rs and antigenicities. However, the recGP5 apparently possesses different N-linked oligosaccharides residues. Its sensitivity to endo-β-galactosidase digestion indicates that poly-N-acetyllactosamine is present on the individually-expressed protein, but not on the authentic GP5 anchored into the virion envelope. The recGP5 apparently accumulates within the ER compartment as a glycoprotein that possesses high-mannose N-linked oligosaccharide side chains sensitive to endo-β-N-acetylglucosaminidase H treatment, by contrast to its viral counterpart for which N-linked oligosaccharide side chains are of both high-mannose and complex types. Coinfection of 293 cells with hAdVs expressing the M and GP5 did not lead to M-GP5 heterodimer formation, as demonstrated in PRRSV-infected cells. Moreover, cells infected with inducible hAdV/ORF5 showed that GP5 of the North American strain is proapoptotic. Indeed, when the expression cassette was turned-on, caspase 3 activity in hAdV/ORF5 infected cells was enhanced and DNA fragmentation could be detected by TUNEL assays. Pigs intradermally injected twice with hAdV/ORF5 developed antibody titers to the authentic viral GP5 as soon as 10 days following challenge with the homologous virulent PRRSV strain, as revealed by Western blot and virus neutralization tests, suggesting the establishment of a specific immune memory.

Biochemical properties and processing of the three major structural proteins of PRRS virus expressed by recombinant adenoviruses. Structural, functional and community aspects.

The ORFs 5 to 7 encode for the three major structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV): the envelope glycoprotein GP5 (25-26 kDa), the non-glycosylated membrane protein M (18-19 kDa) and the nucleocapsid protein N (14-15 kDa), respectively (Mardassi et al., 1995; Meulenberg et al., 1993). The latter structural proteins of PRRSV are closely associated both in the infected-cells and in the virion, the GP5 and M proteins being associated in the form of heterodimers (Mardassi et al., 1996). The GP5, which is highly glycosylated (Mardassi et al., 1996), have been found to play an important role in the induction of a protective immune response (Pirzadeh and Dea, 1998). Immunization experiments of mice with E. coli-expressed GST-ORFS recombinant fusion protein, as well as with purified PRRSV, induced specific anti-GP5 neutra-lizing MAbs (Pirzadeh and Dea, 1997). The antibodies in PRRSV-infected pigs antisera responsible for the viral neutralisation in cell culture have been also determined to be GP5 specific (Gonin et al., 1999). In DNA immunization experiments, pigs injected with a plasmidic vector expressing the ORF5 gene not only produced neutralizing antibodies to PRRSV, but were also protected against development of clinical disease and lung lesions following an intratracheal challenge with a high infectious dose of PRRSV. On the other hand, parenteral inoculation of SPF piglets with E. coli-expressed GST-ORF5 recombinant fusion protein rather resulted in an increased severity of lung lesions, despite the development of high titers (> 2048) of non-neutralizing antibody titers to GP5 (Pirzadeh and Dea, 1998). Consequently, the use of an eucaryotic expression system should be preconized to produce large amounts of a recombinant protein that would have to preserve the major characteristics (glycosylation, conformational epitopes) of the native major envelope glycoprotein of PRRSV. Recently, a replication defective human type 5 adenovirus (Ad) has been used successfully for the eucaryotic expression and characterization of the ORF3 product of PRRSV (Mardassi et al., 1998). Previous investigators have also demonstrated that recombinant Ad (recAd) carrying the structural genes of different viruses, mainly the envelope or spike glycoproteins of murine and porcine coronaviruses, were effective for the induction of antibodies to the expressed rec glycoproteins that conferred protection (Both et al., 1993; Torres et al., 1995; Wesseling et al., 1993).KeywordsRecombinant AdenovirusNucleocapsid ProteinRespiratory Syndrome VirusMajor Structural ProteinSpike GlycoproteinThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Macrophage inflammatory protein-2 gene therapy attenuates adenovirus- and acetaminophen-mediated hepatic injury.

Profound hepatocellular injury is often a consequence of adenovirus-mediated gene therapy or acetaminophen ingestion. The aim of the present study was to examine the role of a CXC chemokine, macrophage inflammatory protein-2 (MIP-2), in the hepatotoxic response by mice infected with adenovirus and challenged with acetaminophen. CD1 mice that received a replication-defective human type 5 adenovirus vector (Ad70-3) intravenously exhibited hepatic injury that peaked at 24 h after infection. In contrast, mice that received a similar adenovirus vector containing a rodent MIP-2 cDNA insert had no hepatic injury at any time after infection. The combination of Ad70-3 infection and an intraperitoneal challenge with 400 mg/kg of acetaminophen was fatal in 50% of the mice, but only 10% of the AdMIP-2 group receiving acetaminophen were similarly affected. Furthermore, AdMIP-2 mice had significantly lower hepatic injury and serum aminotransaminases compared with the Ad70-3 group. However, AdMIP-2 infection in mice lacking the CXC chemokine receptor that binds MIP-2, CXCR2, did not attenuate any of the markers of liver injury after adenovirus and acetaminophen challenge. AdMIP-2 treatment of CD1 mice was also associated with significantly decreased leukocyte infiltration into the liver and an earlier increase in hepatic 3H-thymidine incorporation compared with the control group. Taken together, these data demonstrate that MIP-2 has a protective role in both adenovirus- and acetaminophen-mediated hepatotoxicity, and suggest that MIP-2 may promote rapid hepatic regeneration following acute hepatic injury.

Toxicity of Replication-Defective Adenoviral Recombinants in Dissociated Cultures of Nervous Tissue

Replication-defective human type 5 adenoviral recombinants (AVR) are very efficient means of introducing foreign genes into neuronsin vitroandin vivo;however, a significant reduction in the number of cells expressing reporter genes has been reported to occur over time.In vitro,this may be due to direct toxicity of the protein product of the transgene or adenoviral molecule(s).In vivo,in addition, an immune attack by the host could eliminate the transduced cells. To assess the direct toxicity of AVR or reporter gene products, a quantitative study of survival of transduced neurons over a period of 4 weeks was conducted in primary neural cultures. Cultures of dissociated murine spinal cord–dorsal root ganglia were exposed to AVR containing theEscherichia coli lacZ(E. coli lacZ) gene under control of either the very efficient cytomegalovirus enhancer/promoter or the fast muscle troponin I promoter, which is not active in these cells. Two factors contributed to loss of neuronal and nonneuronal cells: (i) direct toxicity of (E1+E3)-deleted replication-incompetent AVR at high titers [≥5×108viral particles/ml or multiplicity of infection (m.o.i.) 1000] and (ii) high levels of expression of the reporter gene product, β-galactosidase, at titers that result in 55–75% transduction efficiency (5×107–5×108viral particles/ml or m.o.i. 100–1000). Despite the efficacy of adenoviral vectors in introducing foreign genes into primary, postmitotic cells, specific precautions must be taken in their use because of the narrow margin between concentrations of recombinants that transduce a sufficient percentage of cells and those that are cytotoxic.