IntroductionPreventative viral vaccines provide protection through induction of immunologic memory, most notably circulating neutralizing antibodies. 1 For some viruses, such as HIV-1, vaccines have failed to induce protective levels of antibodies and the focus of many of the ongoing HIV-1 vaccine efforts has shifted to T-cell responses. 2 Correlates of T-cell-mediated protection to viral infections remain ill-defined because of the not yet fully understood complexity of memory T-cell responses.Replication-defective adenovirus (Ad) vectors are at the forefront of HIV-1 vaccine research and have entered phase 2 clinical trials. [3][4][5] One of the most remarkable features of Ad-based vaccines is their ability to induce exceptionally high and sustained frequencies of transgene product-specific CD8 ϩ T cells that, unlike those induced by other subunit vaccine carriers such as DNA vaccines or poxvirus vectors, do not contract after the initial activation. 6,7 Here we show that replication-defective E1-deleted Ad vector genomes similar to those of Ads acquired by natural infections 8,9 persist. Persistent vector was found in muscle at the site of inoculation, in liver, and in lymphatic tissues of experimental animals. Within lymphatic tissues the vector genomes are enriched in T-cells directed to the antigen encoded by the viral vector. The vector's genome remains transcriptionally active, and the continued presence of transgene products appears to maintain high frequencies of activated antigen-specific CD8 ϩ T cells in addition to a pool of resting memory T cells. Although the concept of persisting vaccines may provide challenges for their eventual use for mass vaccination, concomitantly maintaining high frequencies of effector-like T cells and resting memory T cells may provide a solution to the dilemma of vaccines that rely on T-cell-mediated protection. Materials and methods MiceC57Bl/6 and BALB/c mice were purchased at 6 to 8 weeks of age from Charles River Laboratories (Boston, MA). OT1 and P14 mice were bred at the Animal Facility of the Wistar Institute (Philadelphia, PA) and typed by polymerase chain reaction (PCR) for homozygosity. Animals were treated according to guidelines of the Wistar Institute. Cell linesHEK 293 and HeLa cells were grown in Dulbecco Modified Eagle medium, supplemented with 10% fetal bovine serum. Viruses and viral vectorsAd vectors expressing Gag of HIV-1, the rabies virus glycoprotein or SIINFEKL as a fusion protein with influenza virus nucleoprotein and green fluorescent protein, the glycoprotein of lymphocytic choriomeningitis virus (LCMV), or green fluorescent protein were propagated on HEK 293 cells, purified, and quality-controlled as described previously. 10 Vaccinia virus vectors expressing Gag were grown on HeLa cells and titrated as described. 11 LCMV strain Armstrong was produced as described. 12 Immunization or infection of miceMice were immunized intramuscularly at 6 to 10 weeks of age with vectors diluted in 100 L PBS. Mice were infected with vaccinia virus vectors or L...

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A universal influenza vaccine, designed to induce broadly cross-reactive immunity against current and future influenza A virus strains, is in critical demand to reduce the need for annual vaccinations with vaccines chosen upon predicting the predominant circulating viral strains, and to ameliorate the threat of cyclically occurring pandemics that have, in the past, killed tens of millions. Here, we describe a vaccine regimen based on sequential immunization with two serologically distinct chimpanzee-derived replication-defective adenovirus (Ad) vectors expressing the matrix-2 protein ectodomain (M2e) from three divergent strains of influenza A virus fused to the influenza virus nucleoprotein (NP) for induction of antibodies to M2e and virus-specific CD8(+) T cells to NP. In preclinical mouse models, the Ad vaccines expressing M2e and NP elicit robust NP-specific CD8(+) T-cell responses and moderate antibody responses to all three M2e sequences. Most importantly, vaccinated mice are protected against morbidity and mortality following challenge with high doses of different influenza virus strains. Protection requires both antibodies to M2e and cellular immune responses to NP.

Adenoviral vectors have shown great promise as vaccine carriers and in gene transfer to correct underlying genetic diseases. Traditionally, construction of adenoviral vectors is complex and time consuming. In this paper, we provide an improved method for efficient generation of novel adenoviral vectors by using direct cloning. We introduce a feasible and detailed protocol for the development of chimpanzee adenoviruses (Ads) as molecular clones, as well as for the generation of recombinant virus from the molecular clones. Recombinant viruses are genetically stable and induce potent immune responses in animals. Generation of new Ad molecular clones or new recombinant Ad can be achieved in 2 months or 2 weeks, respectively.

Using a mouse model we show that self-complementary (sc) adeno-associated virus (AAV) vectors pseudotyped with capsids of serotypes 2, 7 or 8 induce more potent transgene product-specific CD8(+) T cell and antibody responses compared to corresponding single-stranded (ss)AAV vectors. These data suggest that the higher and more rapidly appearing amounts of transgene product achieved with scAAV vectors may increase detrimental immune responses in gene transfer recipients.