An adenovirus previously isolated from a mesenteric lymph node from a chimpanzee was fully sequenced and found to be similar in overall structure to human adenoviruses. The genome of this virus, called C68, is 36,521 bp in length and is most similar to subgroup E of human adenovirus, with 90% identity in most adenovirus type 4 open reading frames that have been sequenced. Substantial differences in the hexon hypervariable regions were noted between C68 and other known adenoviruses, including adenovirus type 4. Neutralizing antibodies to C68 were highly prevalent in sera from a population of chimpanzees, while sera from humans and rhesus monkeys failed to neutralize C68. Furthermore, infection with C68 was not neutralized from sera of mice immunized with human adenovirus serotypes 2, 4, 5, 7, and 12. A replication-defective version of C68 was created by replacing the E1a and E1b genes with a minigene cassette; this vector was efficiently transcomplemented by the E1 region of human adenovirus type 5. C68 vector transduced a number of human and murine cell lines. This nonhuman adenoviral vector is sufficiently similar to human serotypes to allow growth in 293 cells and transduction of cells expressing the coxsackievirus and adenovirus receptor. As it is dissimilar in regions such as the hexon hypervariable domains, C68 vector avoids significant cross-neutralization by sera directed against human serotypes.Vectors based on human adenovirus subgroup C (i.e., types 2 and 5) have realized widespread application in preclinical and clinical models of gene therapy (34). The viruses are rendered replication defective by deletion of E1 sequences. Multiple essential genes are disabled in more advanced versions of adenovirus vectors (7,10,17,31). An important limitation of the use of adenovirus type 2-and adenovirus type 5-based vectors for human applications is that many individuals are immune to the virus as the result of a previous natural infection (6). A manifestation of existing immunity to the virus is B-cell activation, leading to persistent neutralizing antibodies that block vector uptake in vivo and diminish transduction.One approach to accomplish immunologic distinction is to engineer the capsid of an adenovirus type 5-or adenovirus type 2-based vector. Several studies have attempted to accomplish this by exchanging the gene encoding fiber, since the protein is directly involved in receptor binding. While this has been successful in redirecting uptake of vector via a pathway distinct from that directed by the coxsackievirus and adenovirus (CAR) receptor, such chimeric viruses are still cross-neutralized due to blocking antibodies directed against hexon epitopes in the hypervariable regions (11,14,19,28,31). Recent attempts to engineer hexon proteins in chimeric viruses have been complicated by serotype-specific constraints in the hexon structure, which compromise the formation of stable chimeras. Selective modification of the hypervariable regions of hexon have diminished type-specific cross-neutralization in vitro...

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...

In animal models, E1-deleted human adenoviral recombinants of the serotype 5 (AdHu5) have shown high efficacy as vaccine carriers for different Ags including those of HIV-1. Humans are infected by common serotypes of human adenovirus such as AdHu5 early in life and a significant percentage has high levels of neutralizing Abs to these serotypes, which will very likely impair the efficacy of recombinant vaccines based on the homologous virus. To circumvent this problem, a novel replication-defective adenoviral vaccine carrier based on an E1-deleted recombinant of the chimpanzee adenovirus 68 (AdC68) was developed. An AdC68 construct expressing a codon-optimized, truncated form of gag of HIV-1 induces CD8+ T cells to gag in mice which at the height of the immune response encompass nearly 20% of the entire splenic CD8+ T cell population. The vaccine-induced immune response provides protection to challenge with a vaccinia gag recombinant virus. Induction of transgene-specific CD8+ T cells and protection against viral challenge elicited by the AdC68 vaccines is not strongly inhibited in animals preimmune to AdHu5 virus. However, the response elicited by the AdHu5 vaccine is greatly attenuated in AdHu5 preimmune animals.

In this manuscript, an E1 and E3 deleted adenoviral recombinant expressing the rabies virus glycoprotein (G protein) under the control of the cytomegalovirus early promoter was tested for induction of a rabies virus-specific immune response in mice. The construct was found to induce neutralizing antibodies and cytolytic T cells to rabies virus. Mice vaccinated with the adenoviral construct either by the systemic route or by application into the airways were protected against a subsequent infection with a virulent strain of rabies virus. The efficacy of the replication-defective construct was far superior to that of a well-characterized vaccinia rabies glycoprotein recombinant.

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The oral cavity, as the entry point to the body, may play a critical role in the pathogenesis of SARS-CoV-2 infection that has caused a global outbreak of the coronavirus disease 2019 (COVID-19). Available data indicate that the oral cavity may be an active site of infection and an important reservoir of SARS-CoV-2. Considering that the oral surfaces are colonized by a diverse microbial community, it is likely that viruses have interactions with the host microbiota. Patients infected by SARS-CoV-2 may have alterations in the oral and gut microbiota, while oral species have been found in the lung of COVID-19 patients. Furthermore, interactions between the oral, lung, and gut microbiomes appear to occur dynamically whereby a dysbiotic oral microbial community could influence respiratory and gastrointestinal diseases. However, it is unclear whether SARS-CoV-2 infection can alter the local homeostasis of the resident microbiota, actively cause dysbiosis, or influence cross-body sites interactions. Here, we provide a conceptual framework on the potential impact of SARS-CoV-2 oral infection on the local and distant microbiomes across the respiratory and gastrointestinal tracts ('oral-tract axes'), which remains largely unexplored. Studies in this area could further elucidate the pathogenic mechanism of SARS-CoV-2 and the course of infection as well as the clinical symptoms of COVID-19 across different sites in the human host.

A plasmid vector expressing the full-length rabies virus glycoprotein (G protein) under the control of the simian virus 40 (SV40) promoter has previously been shown to induce upon intramuscular (i.m.) inoculation into mice a specific B- and T-cell-mediated immune response and protection against challenge with a virulent strain of the virus. Here we tested two parameters that might affect the efficacy of this DNA vaccine. First, we replaced the SV40 promoter of the original vector with the early promoter derived from cytomegalovirus leaving all other parameters of the plasmid intact. Although upon transfection in vitro the two vectors showed a striking difference in their ability to cause stable expression of the rabies virus G protein, upon i.m. inoculation into mice both constructs induced comparable immune responses. Second, we constructed a vector that induces expression of a secreted form of rabies G protein by inserting a stop codon just upstream of the transmembrane domain of the rabies G protein gene. The immune responses to the DNA vaccines expressing the two different forms of the G protein, secreted and membrane bound, were compared and found to be similar in magnitude. The long-term effect of DNA vaccination was also investigated especially with regard to adverse immunological reactions such as the induction of unresponsiveness against rabies virus and the development of antibodies to DNA. DNA vaccination was found to induce long-lasting immunity to rabies virus without apparent negative side effects such as development of T cell tolerance or generation of anti-DNA antibodies.