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Unlabelled: Induction of long-lasting immunity against viral respiratory tract infections remains an elusive goal. Using a nonhuman primate model of human respiratory syncytial virus (hRSV) infection, we compared mucosal and systemic immune responses induced by different DNA delivery approaches to a novel parenteral DNA prime-tonsillar adenoviral vector booster immunization regimen. Intramuscular (i.m.) electroporation (EP) of a DNA vaccine encoding the fusion protein of hRSV induced stronger systemic immune responses than intradermal EP, tattoo immunization, and conventional i.m. DNA injection. A single EP i.m., followed by two atraumatic tonsillar immunizations with the adenoviral vector, elicited strong systemic immune responses, an unique persistent CD4(+) and CD8(+) T cell response in the lower respiratory tract and protection from intranasal hRSV challenge. Thus, parenteral DNA priming followed by booster immunization targeted to a mucosal inductive site constitutes an effective vaccine regimen for eliciting protective immune responses at mucosal effector sites., Importance: The human respiratory syncytial virus (hRSV) is the most common cause of severe respiratory tract disease in infancy and leads to substantial morbidity and morality in the elderly. In this study, we compared the immunogenicity and efficacy of several gene-based immunization protocols in rhesus macaques. Thereby, we found that the combination of an initially parenterally delivered DNA vaccine with a subsequent atraumatic tonsillar adenoviral vector immunization results in a strong systemic immune response accompanied by an exceptional high T-cell response in the mucosa. Strikingly, these animals were protected against a RSV challenge infection controlling the viral replication indicated by a 1,000-fold-lower viral load in the lower respiratory tract. Since mucosal cellular responses of this strength had not been described in earlier RSV vaccine studies, this heterologous DNA prime-tonsillar boost vaccine strategy is very promising and should be pursued for further preclinical and clinical testing.

To explore the efficacy of novel complementary prime-boost immunization regimens in a nonhuman primate model for HIV infection, rhesus monkeys primed by different DNA vaccines were boosted with virus-like particles (VLP) and then challenged by repeated low-dose rectal exposure to simian immunodeficiency virus (SIV). Characteristic of the cellular immune response after the VLP booster immunization were high numbers of SIV-specific, gamma interferon-secreting cells after stimulation with inactivated SIV particles, but not SIV peptides, and the absence of detectable levels of CD8(+) T cell responses. Antibodies specific to SIV Gag and SIV Env could be induced in all animals, but, consistent with a poor neutralizing activity at the time of challenge, vaccinated monkeys were not protected from acquisition of infection and did not control viremia. Surprisingly, vaccinees with high numbers of SIV-specific, gamma interferon-secreting cells were infected fastest during the repeated low-dose exposures and the numbers of these immune cells in vaccinated macaques correlated with susceptibility to infection. Thus, in the absence of protective antibodies or cytotoxic T cell responses, vaccine-induced immune responses may increase the susceptibility to acquisition of immunodeficiency virus infection. The results are consistent with the hypothesis that virus-specific T helper cells mediate this detrimental effect and contribute to the inefficacy of past HIV vaccination attempts (e.g., STEP study).

In some retroviruses, such as Rous sarcoma virus and prototype foamy virus, Gag proteins are known to shuttle between the nucleus and the cytoplasm and are implicated in nuclear export of the viral genomic unspliced RNA (gRNA) for subsequent encapsidation. A similar function has been proposed for human immunodeficiency virus type 1 (HIV-1) Gag based on the identification of nuclear localization and export signals. However, the ability of HIV-1 Gag to transit through the nucleus has never been confirmed. In addition, the lentiviral Rev protein promotes efficient nuclear gRNA export, and previous reports indicate a cytoplasmic interaction between Gag and gRNA. Therefore, functional effects of HIV-1 Gag on gRNA and its usage were explored. Expression of gag in the absence of Rev was not able to increase cytoplasmic gRNA levels of subgenomic, proviral, or lentiviral vector constructs, and gene expression from genomic reporter plasmids could not be induced by Gag provided in trans. Furthermore, Gag lacking the reported nuclear localization and export signals was still able to mediate an efficient packaging process. Although small amounts of Gag were detectable in the nuclei of transfected cells, a Crm1-dependent nuclear export signal in Gag could not be confirmed. Thus, our study does not provide any evidence for a nuclear function of HIV-1 Gag. The encapsidation process of HIV-1 therefore clearly differs from that of Rous sarcoma virus and prototype foamy virus.

Processing and presentation of vaccine antigens by professional antigen-presenting cells (APCs) is of great importance for the efficient induction of protective immunity. We analyzed whether the efficacy of an adenovirus-based retroviral vaccine can be enhanced by coadministration of adenovirus-encoded chemokines that attract and stimulate APCs. In the Friend retrovirus (FV) mouse model we coexpressed CCL3, CCL20, CCL21, or CXCL14 from adenoviral vectors, together with FV Gag and Env antigens, and then analyzed immune responses and protection from pathogenic FV infection. Although most tested chemokines did not improve protection against FV challenge, mice that received adenoviral vectors encoding CCL3 together with FV antigens showed significantly better control over viral loads and FV-induced disease than mice immunized with the viral antigens only. Improved protection correlated with enhanced virus-specific CD4+ T cell responses and higher neutralizing antibody titers. To apply these results to an HIV vaccine, mice were immunized with adenoviral vectors encoding the HIV antigens Env and Gag-Pol and coadministered vectors encoding CCL3. Again, this combination vaccine induced higher virus-specific antibody titers and CD4+ T cell responses than did the HIV antigens alone. These results indicate that coexpression of the chemokine CCL3 by adenovirus-based vectors may be a promising tool to improve antiretroviral vaccination strategies.

Although the viral Rev protein is necessary for HIV replication, its main function in the viral replication cycle has been controversial. Reinvestigating the effect of Rev on the HIV-1 RNA distribution in various cell lines and primary cells revealed that Rev enhanced cytoplasmic levels of the unspliced HIV-1 RNA, mostly 3- to 12-fold, while encapsidation of the RNA and viral infectivity could be stimulated >1,000-fold. Although this clearly questions the general notion that the nuclear export of viral RNAs is the major function of Rev, mechanistically encapsidation seems to be linked to nuclear export, since the tethering of the nuclear export factor TAP to the HIV-1 RNA also enhanced encapsidation. Interference with the formation of an inhibitory ribonucleoprotein complex in the nucleus could lead to enhanced accessibility of the cytoplasmic HIV-1 RNA for translation and encapsidation. This might explain why Rev and tethered TAP exert the same pattern of pleiotropic effects.

We present a new type of adenoviral vector that both encodes and displays a vaccine antigen on the capsid, thus combining in itself gene-based and protein vaccination; this vector resulted in an improved vaccination outcome in the Friend virus (FV) model. For presentation of the envelope protein gp70 of Friend murine leukemia virus on the adenoviral capsid, gp70 was fused to the adenovirus capsid protein IX. When compared to vaccination with conventional FV Env- and Gag-encoding adenoviral vectors, vaccination with the adenoviral vector that encodes and displays pIX-gp70 combined with an FV Gag-encoding vector resulted in significantly improved protection against systemic FV challenge infection, with highly controlled viral loads in plasma and spleen. This improved protection correlated with improved neutralizing antibody titers and stronger CD4(+) T-cell responses. Using a vector that displays gp70 without encoding it, we found that while the antigen display on the capsid alone was sufficient to induce high levels of binding antibodies, in vivo expression was necessary for the induction of neutralizing antibodies. This new type of adenovirus-based vaccine could be a valuable tool for vaccination.

Adenoviral vectors (AdV) have received considerable attention for vaccine development because of their high immunogenicity and efficacy. In previous studies, it was shown that DNA immunization of mice with codon-optimized expression plasmids encoding the fusion protein of respiratory syncytial virus (RSV F) resulted in enhanced protection against RSV challenge compared to immunization with plasmids carrying the wild-type cDNA sequence of RSV F. In this study, we constructed AdV carrying the codon-optimized full-length RSV F gene (AdV-F) or the soluble form of the RSV F gene (AdV-Fsol). BALB/c mice were immunized twice with AdV-F or AdV-Fsol and challenged with RSV intranasally. Substantial levels of antibody to RSV F were induced by both AdV vaccines, with peak neutralizing-antibody titers of 1:900. Consistently, the viral loads in lung homogenates and bronchoalveolar lavage fluids were significantly reduced by a factor of more than 60,000. The protection against viral challenge could be measured even 8 months after the booster immunization. AdV-F and AdV-Fsol induced similar levels of immunogenicity and protective efficacy. Therefore, these results encourage further development of AdV vaccines against RSV infection in humans.

During cell culture isolation experiments to recover Dobrava hantavirus from a suspension of liver from a striped field mouse (Apodemus agrarius), an unknown virus was coisolated. Atypically for hantaviruses, it had extensive cytopathic effects. Using a random PCR approach, it was identified as a novel murine adenovirus, MAdV-3 (for MAdV type 3). A plaque-purified virus clone was prepared and further characterized. The complete genome sequence of MAdV-3 was determined to be 30,570 bp in length. Sequence comparisons to other adenovirus species revealed highest similarity to MAdV-1, the representative of the murine adenovirus A species. However, substantial differences were found in the E1, E3, and E4 genomic regions. The phylogenetic distance of MAdV-3 amino acid sequences for pVIII, protease, polymerase, and hexon from MAdV-1 is markedly higher than 0.1 exchange per position, and, based on our cross-neutralization experiments, MAdV-3 and MAdV-1 can be regarded as different serotypes. Therefore, we propose to classify MAdV-3 as the first isolate of a novel adenovirus species, designated murine adenovirus C (MAdV-C). The novel MAdV-3 virus is not only genetically and serologically distinct from MAdV-1 but also shows a unique organ tropism in infected mice. In contrast to MAdV-1, the virus was not detectable in brain but predominantly infected heart tissue. Thus, infection of mice with cardiotropic MAdV-3 might be an interesting animal model of adenovirus-induced myocarditis.

The development of needle-free vaccines is one of the recently defined "grand challenges in global health" (H. Varmus, R. Klausner, R. Klausner, R. Zerhouni, T. Acharya, A. S. Daar, and P. A. Singer, Science 302:398-399, 2003). To explore whether a natural pathway to the inductive site of the mucosa-associated lymphatic tissue could be exploited for atraumatic immunization purposes, replication-deficient viral vector vaccines were sprayed directly onto the tonsils of rhesus macaques. Tonsillar immunization with viral vector vaccines encoding simian immunodeficiency virus (SIV) antigens induced cellular and humoral immune responses. Viral RNA levels after a stringent SIV challenge were reduced, providing a level of protection similar to that observed after systemic immunization with the same vaccines. Thus, atraumatic oral spray immunization with replication-deficient vectors can overcome the epithelial barrier, deliver the vaccine antigen to the mucosa-associated lymphatic tissue, and avoid induction of tolerance, providing a novel approach to circumvent acceptability problems of syringe and needle vaccines for children and in developing countries.

Preferential integration into transcriptionally active regions of genomes has been observed for retroviral vectors based on gamma-retroviruses and lentiviruses. However, differences in the integration site preferences were detected, which might be explained by differences in viral components of the preintegration complexes. Viral determinants of integration site preferences have not been defined. Therefore, integration sites of simian immunodeficiency virus (SIV)-based vectors produced in the absence of accessory genes or lacking promoter and enhancer elements were compared. Similar integration patterns for the different SIV vectors indicate that vif, vpr, vpx, nef, env, and promoter or enhancer elements are not required for preferential integration of SIV into transcriptionally active regions of genomes.

Oncolytic adenoviruses have emerged as a promising approach for the treatment of tumors resistant to other treatment modalities. However, preclinical safety studies are hampered by the lack of a permissive nonhuman host. Screening of a panel of primary cell cultures from seven different animal species revealed that porcine cells support productive replication of human adenovirus type 5 (Ad5) nearly as efficiently as human A549 cells, while release of infectious virus by cells from other animal species tested was diminished by several orders of magnitude. Restriction of productive Ad5 replication in rodent and rabbit cells seems to act primarily at a postentry step. Replication efficiency of adenoviral vectors harboring different E1 deletions or mutations in porcine cells was similar to that in A549 cells. Side-by-side comparison of the viral load kinetics in blood of swine and mice injected with Ad5 or a replication-deficient adenoviral vector failed to provide clear evidence for virus replication in mice. In contrast, evidence suggests that adenovirus replication occurs in swine, since adenoviral late gene expression produced a 13.5-fold increase in viral load in an individual swine from day 3 to day 7 and 100-fold increase in viral DNA levels in the Ad5-infected swine compared to the animal receiving a replication-deficient adenovirus. Lung histology of Ad5-infected swine revealed a severe interstitial pneumonia. Although the results in swine are based on a small number of animals and need to be confirmed, our data strongly suggest that infection of swine with human adenovirus or oncolytic adenoviral vectors is a more appropriate animal model to study adenoviral pathogenicity or pharmacodynamic and toxicity profiles of adenoviral vectors than infection of mice.

Infection of cells transduced with a lentiviral vector by human immunodeficiency virus (HIV) could lead to packaging of the lentiviral vector RNA into HIV particles and unintended transfer of the vector. To prevent this, the Rev-responsive element (RRE) of an HIV-1 vector was functionally replaced by a heterologous RNA element (MS2). Providing Rev fused to an MS2 binding protein allowed efficient vector production. Mobilization of the vector from infected target cells was below the level of detection and at least 10(3)- to 10(4)-fold lower than for the RRE-containing vector. Thus, RRE-deficient lentiviral vectors provide a novel approach to reduce the risk of vector mobilization.

The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes severe pneumonia with a fatal outcome in approximately 10% of patients. SARS-CoV is not closely related to other coronaviruses but shares a similar genome organization. Entry of coronaviruses into target cells is mediated by the viral S protein. We functionally analyzed SARS-CoV S using pseudotyped lentiviral particles (pseudotypes). The SARS-CoV S protein was found to be expressed at the cell surface upon transient transfection. Coexpression of SARS-CoV S with human immunodeficiency virus-based reporter constructs yielded viruses that were infectious for a range of cell lines. Most notably, viral pseudotypes harboring SARS-CoV S infected hepatoma cell lines but not T- and B-cell lines. Infection of the hepatoma cell line Huh-7 was also observed with replication-competent SARS-CoV, indicating that hepatocytes might be targeted by SARS-CoV in vivo. Inhibition of vacuolar acidification impaired infection by SARS-CoV S-bearing pseudotypes, indicating that S-mediated entry requires low pH. Finally, infection by SARS-CoV S pseudotypes but not by vesicular stomatitis virus G pseudotypes was efficiently inhibited by a rabbit serum raised against SARS-CoV particles and by sera from SARS patients, demonstrating that SARS-CoV S is a target for neutralizing antibodies and that such antibodies are generated in SARS-CoV-infected patients. Our results show that viral pseudotyping can be employed for the analysis of SARS-CoV S function. Moreover, we provide evidence that SARS-CoV infection might not be limited to lung tissue and can be inhibited by the humoral immune response in infected patients.

Deletion of the nef gene from simian immunodeficiency virus (SIV) strain SIVmac239 yields a virus that undergoes attenuated growth in rhesus macaques and offers substantial protection against a subsequent challenge with some SIV wild-type viruses. We used a recently described model to identify sites in which the SIVDeltanef vaccine strain replicates and elicits immunity in vivo. A high dose of SIVDeltanef was applied to the palatine and lingual tonsils, where it replicated vigorously in this portal of entry at 7 days. Within 2 weeks, the virus had spread and was replicating actively in axillary lymph nodes, primarily in extrafollicular T-cell-rich regions but also in germinal centers. At this time, large numbers of perforin-positive cells, both CD8(+) T cells and CD3-negative presumptive natural killer cells, were found in the tonsil and axillary lymph nodes. The number of infected cells and perforin-positive cells then fell. When autopsy studies were carried out at 26 weeks, only 1 to 3 cells hybridized for viral RNA per section of lymphoid tissue. Nevertheless, infected cells were detected chronically in most lymphoid organs, where the titers of infectious virus could exceed by a log or more the titers in blood. Immunocytochemical labeling at the early active stages of infection showed that cells expressing SIVDeltanef RNA were CD4(+) T lymphocytes. A majority of infected cells were not in the active cell cycle, since 60 to 70% of the RNA-positive cells in tissue sections lacked the Ki-67 cell cycle antigen, and both Ki-67-positive and -negative cells had similar grain counts for viral RNA. Macrophages and dendritic cells, identified with a panel of monoclonal antibodies to these cells, were rarely infected. We conclude that the attenuated growth and protection observed with the SIVDeltanef vaccine strain does not require that the virus shift its characteristic site of replication, the CD4(+) T lymphocyte. In fact, this immunodeficiency virus can replicate actively in CD4(+) T cells prior to being contained by the host, at least in part by a strong killer cell response that is generated acutely in the infected lymph nodes.