The 2000HIV study: Design, multi-omics methods and participant characteristics.

Background: Even during long-term combination antiretroviral therapy (cART), people living with HIV (PLHIV) have a dysregulated immune system, characterized by persistent immune activation, accelerated immune ageing and increased risk of non-AIDS comorbidities. A multi-omics approach is applied to a large cohort of PLHIV to understand pathways underlying these dysregulations in order to identify new biomarkers and novel genetically validated therapeutic drugs targets. Methods: The 2000HIV study is a prospective longitudinal cohort study of PLHIV on cART. In addition, untreated HIV spontaneous controllers were recruited. In-depth multi-omics characterization will be performed, including genomics, epigenomics, transcriptomics, proteomics, metabolomics and metagenomics, functional immunological assays and extensive immunophenotyping. Furthermore, the latent viral reservoir will be assessed through cell associated HIV-1 RNA and DNA, and full-length individual proviral sequencing on a subset. Clinical measurements include an ECG, carotid intima-media thickness and plaque measurement, hepatic steatosis and fibrosis measurement as well as psychological symptoms and recreational drug questionnaires. Additionally, considering the developing pandemic, COVID-19 history and vaccination was recorded. Participants return for a two-year follow-up visit. The 2000HIV study consists of a discovery and validation cohort collected at separate sites to immediately validate any finding in an independent cohort. Results: Overall, 1895 PLHIV from four sites were included for analysis, 1559 in the discovery and 336 in the validation cohort. The study population was representative of a Western European HIV population, including 288 (15.2%) cis-women, 463 (24.4%) non-whites, and 1360 (71.8%) MSM (Men who have Sex with Men). Extreme phenotypes included 114 spontaneous controllers, 81 rapid progressors and 162 immunological non-responders. According to the Framingham score 321 (16.9%) had a cardiovascular risk of >20% in the next 10 years. COVID-19 infection was documented in 234 (12.3%) participants and 474 (25.0%) individuals had received a COVID-19 vaccine. Conclusion: The 2000HIV study established a cohort of 1895 PLHIV that employs multi-omics to discover new biological pathways and biomarkers to unravel non-AIDS comorbidities, extreme phenotypes and the latent viral reservoir that impact the health of PLHIV. The ultimate goal is to contribute to a more personalized approach to the best standard of care and a potential cure for PLHIV.

Adenovirus vector-based multi-epitope vaccine provides partial protection against H5, H7, and H9 avian influenza viruses.

The emergence of H5, H7, and H9 avian influenza virus subtypes in humans reveals their pandemic potential. Although human-to-human transmission has been limited, the genetic reassortment of the avian and human/porcine influenza viruses or mutations in some of the genes resulting in virus replication in the upper respiratory tract of humans could generate novel pandemic influenza viruses. Current vaccines do not provide cross protection against antigenically distinct strains of the H5, H7, and H9 influenza viruses. Therefore, newer vaccine approaches are needed to overcome these potential threats. We developed an egg-independent, adenovirus vector-based, multi-epitope (ME) vaccine approach using the relatively conserved immunogenic domains of the H5N1 influenza virus [M2 ectodomain (M2e), hemagglutinin (HA) fusion domain (HFD), T-cell epitope of nucleoprotein (TNP). and HA α-helix domain (HαD)]. Our ME vaccine induced humoral and cell-mediated immune responses and caused a significant reduction in the viral loads in the lungs of vaccinated mice that were challenged with antigenically distinct H5, H7, or H9 avian influenza viruses. These results suggest that our ME vaccine approach provided broad protection against the avian influenza viruses. Further improvement of this vaccine will lead to a pre-pandemic vaccine that may lower morbidity, hinder transmission, and prevent mortality in a pandemic situation before a strain-matched vaccine becomes available.

Vaccine approaches conferring cross-protection against influenza viruses.

INTRODUCTION: Annual vaccination is one of the most efficient and cost-effective strategies to prevent and control influenza epidemics. Most of the currently available influenza vaccines are strong inducers of antibody responses against viral surface proteins, hemagglutinin (HA) and neuraminidase (NA), but are poor inducers of cell-mediated immune responses against conserved internal proteins. Moreover, due to the high variability of viral surface proteins because of antigenic drift or antigenic shift, many of the currently licensed vaccines confer little or no protection against drift or shift variants. Areas covered: Next generation influenza vaccines that can induce humoral immune responses to receptor-binding epitopes as well as broadly neutralizing conserved epitopes, and cell-mediated immune responses against highly conserved internal proteins would be effective against variant viruses as well as a novel pandemic influenza until circulating strain-specific vaccines become available. Here we discuss vaccine approaches that have the potential to provide broad spectrum protection against influenza viruses. Expert commentary: Based on current progress in defining cross-protective influenza immunity, it seems that the development of a universal influenza vaccine is feasible. It would revolutionize the strategy for influenza pandemic preparedness, and significantly impact the shelf-life and protection efficacy of seasonal influenza vaccines.

Adenoviral E4 34K protein interacts with virus packaging components and may serve as the putative portal.

Studies on dsDNA bacteriophages have revealed that a DNA packaging complex assembles at a special vertex called the 'portal vertex' and consists of a portal, a DNA packaging ATPase and other components. AdV protein IVa2 is presumed to function as a DNA packaging ATPase. However, a protein that functions as a portal is not yet identified in AdVs. To identify the AdV portal, we performed secondary structure analysis on a set of AdV proteins and compared them with the clip region of the portal proteins of bacteriophages phi29, SPP1 and T4. Our analysis revealed that the E4 34K protein of HAdV-C5 contains a region of strong similarity with the clip region of the known portal proteins. E4 34K was found to be present in empty as well as mature AdV particles. In addition, E4 34K co-immunoprecipitates and colocalizes with AdV packaging proteins. Immunogold electron microscopy demonstrated that E4 34K is located at a single site on the virus surface. Finally, tertiary structure prediction of E4 34K and its comparison with that of single subunits of Phi29, SPP1 and T4 portal proteins revealed remarkable similarity. In conclusion, our results suggest that E4 34K is the putative AdV portal protein.

155R is a novel structural protein of bovine adenovirus type 3, but it is not essential for virus replication.

Bovine adenovirus (AdV) type 3 (BAdV-3) E1 region shares functional homology with E1 of human AdV type C5. Sequence analysis of the BAdV-3 E1 region revealed the presence of a novel 155R ORF that is not observed in other AdVs, on the lower strand antiparallel to a portion of the E1B region. The 155R gene products in BAdV-3-infected cells were identified by Northern blot, reverse transcriptase PCR followed by sequencing and Western blot analysis using the155R-specific antibody. 155R seems to be a late protein and is present in purified BAdV-3 particles. Replication kinetics of BAdV mutants with either one (BAdV/155R/mt1) or two (BAdV/155R/mt2) stop codons in the 155R ORF were comparable to those of BAdV-3, indicating that 155R is not essential for virus replication in cell culture. These results suggest that 155R-deleted BAdV-3 vectors could be generated in a cell line that fully complements BAdV-3 E1 functions.

Adenoviral L4 33K forms ring-like oligomers and stimulates ATPase activity of IVa2: implications in viral genome packaging.

The mechanism of genome packaging in adenoviruses (AdVs) is presumed to be similar to that of dsDNA viruses including herpesviruses and dsDNA phages. First, the empty capsids are assembled after which the viral genome is pushed through a unique vertex by a motor which consists of three minimal components: an ATPase, a small terminase and a portal. Various components of this motor exist as ring-like structures forming a central channel through which the DNA travels during packaging. In AdV, the IVa2 protein is believed to function as a packaging ATPase, however, the equivalents of the small terminase and the portal have not been identified in AdVs. IVa2 interacts with another viral protein late region 4 (L4) 33K which is important for genome packaging. Both IVa2 and 33K are expressed at high levels during the late stage of virus infection. The oligomeric state of IVa2 and 33K was analyzed in virus-infected cells, IVa2 and 33K transfected cells, AdV particles, or as recombinant purified proteins. Electron microscopy of the purified proteins showed ring-like oligomers for both proteins which is consistent with their putative roles as a part of the packaging motor. We found that the ATPase activity of IVa2 is stimulated in the presence of 33K and the AdV genome. Our results suggest that the 33K functions analogous to the small terminase proteins and so will be part of the packaging motor complex.

Beta-defensin 2 enhances immunogenicity and protection of an adenovirus-based H5N1 influenza vaccine at an early time.

Reports of human infections with highly pathogenic H5N1 avian influenza viruses in many countries in Asia and Africa with varying case fatality rates highlight the pandemic potential of these viruses. In order to contain a rapidly spreading influenza virus in a pandemic scenario, a vaccine which can induce rapid and robust immune responses, preferably in a single dose, is necessary. Murine beta-defensin 2 (Mbd2), a small molecular weight protein expressed by epithelial cells, has been shown to enhance antigen-specific immune responses by recruiting and activating professional antigen presenting cells to the site of vaccination. This study assessed the potential of Mbd2 to enhance the immunogenicity and protective efficacy of a human adenovirus (HAd)-based vaccine expressing the hemagglutinin (HA) and nucleoprotein (NP) [HAd-HA-NP] of an H5N1 influenza virus. A single inoculation of mice with both HAd-HA-NP and a HAd vector expressing Murine ß-defensin 2 (HAd-Mbd2) resulted in significantly higher levels of both humoral and cell-mediated immune responses compared to the groups vaccinated only with HAd-HA-NP. These responses were evident even at day 7 post-immunization. Furthermore, the HAd-HA-NP+HAd-Mbd2-immunized group receiving the lowest vector dose (2 × 10(7)+1 × 10(7)) was completely protected against an rgH5N1 virus challenge on day 7 post-vaccination. These results highlight the potential of Mbd2 as a genetic adjuvant in inducing rapid and robust immune responses to a HAd-based vaccine.

Adenoviral vector expressing murine ß-defensin 2 enhances immunogenicity of an adenoviral vector based H5N1 influenza vaccine in aged mice.

The ability to resist infections and respond to vaccinations is greatly reduced in the older adult population owing to a general decline in innate and adaptive immune functions with aging. Over the years several strategies such as increasing the vaccine dose, number of immunizations and using adjuvants have been evaluated to improve the immunogenicity and efficacy of vaccines in the older adult population. Murine ß-defensin 2 (Mbd2) has been shown to function as a molecular adjuvant by recruiting and activating immature dendritic cells (DCs), professional antigen-presenting cells (APC), to the site of the immunization. In this study, we evaluated the potential utility of Mbd2 to enhance the efficacy of an adenoviral vector-based H5N1 influenza vaccine expressing hemagglutinin (HA) and nucleoprotein (NP) (HAd-HA-NP) in an aged mouse model. Our results indicated that immunostimulation with an adenoviral vector expressing Mbd2 (HAd-Mbd2) activated DCs and significantly enhanced the humoral and cellular immune responses induced by HAd-HA-NP. Furthermore, immunostimulation with HAd-Mbd2 followed by immunization with HAd-HA-NP resulted in significantly lower virus titers in the lungs following challenge with a H5N1 influenza virus compared to the group immunized with HAd-HA-NP without immunostimulation. Overall, our results highlight the potential utility of Mbd2 as a molecular adjuvant to enhance the immunogenicity and protective efficacy of vaccines for the elderly.

Broadly protective adenovirus-based multivalent vaccines against highly pathogenic avian influenza viruses for pandemic preparedness.

Recurrent outbreaks of H5, H7 and H9 avian influenza viruses in domestic poultry accompanied by their occasional transmission to humans have highlighted the public health threat posed by these viruses. Newer vaccine approaches for pandemic preparedness against these viruses are needed, given the limitations of vaccines currently approved for H5N1 viruses in terms of their production timelines and the ability to induce protective immune responses in the absence of adjuvants. In this study, we evaluated the feasibility of an adenovirus (AdV)-based multivalent vaccine approach for pandemic preparedness against H5, H7 and H9 avian influenza viruses in a mouse model. Replication-defective AdV vectors expressing hemagglutinin (HA) from different subtypes and nucleoprotein (NP) from one subtype induced high levels of humoral and cellular immune responses and conferred protection against virus replication following challenge with H5, H7 and H9 avian influenza virus subtypes. Inclusion of HA from the 2009 H1N1 pandemic virus in the vaccine formulation further broadened the vaccine coverage. Significantly high levels of HA stalk-specific antibodies were observed following immunization with the multivalent vaccine. Inclusion of NP into the multivalent HA vaccine formulation resulted in the induction of CD8 T cell responses. These results suggest that a multivalent vaccine strategy may provide reasonable protection in the event of a pandemic caused by H5, H7, or H9 avian influenza virus before a strain-matched vaccine can be produced.

Adenoviral E2 IVa2 protein interacts with L4 33K protein and E2 DNA-binding protein.

Adenovirus (AdV) is thought to follow a sequential assembly pathway similar to that observed in dsDNA bacteriophages and herpesviruses. First, empty capsids are assembled, and then the genome is packaged through a ring-like structure, referred to as a portal, located at a unique vertex. In human AdV serotype 5 (HAdV5), the IVa2 protein initiates specific recognition of viral genome by associating with the viral packaging domain located between nucleotides 220 and 400 of the genome. IVa2 is located at a unique vertex on mature capsids and plays an essential role during genome packaging, most likely by acting as a DNA packaging ATPase. In this study, we demonstrated interactions among IVa2, 33K and DNA-binding protein (DBP) in virus-infected cells by in vivo cross-linking of HAdV5-infected cells followed by Western blot, and co-immunoprecipitation of IVa2, 33K and DBP from nuclear extracts of HAdV5-infected cells. Confocal microscopy demonstrated co-localization of IVa2, 33K and DBP in virus-infected cells and also in cells transfected with IVa2, 33K and DBP genes. Immunogold electron microscopy of purified HAdV5 showed the presence of IVa2, 33K or DBP at a single site on the virus particles. Our results provide indirect evidence that IVa2, 33K and DBP may form a complex at a unique vertex on viral capsids and cooperate in genome packaging.

Impact of preexisting adenovirus vector immunity on immunogenicity and protection conferred with an adenovirus-based H5N1 influenza vaccine.

The prevalence of preexisting immunity to adenoviruses in the majority of the human population might adversely impact the development of adaptive immune responses against adenovirus vector-based vaccines. To address this issue, we primed BALB/c mice either intranasally (i.n.) or intramuscularly (i.m.) with varying doses of wild type (WT) human adenovirus subtype 5 (HAd5). Following the development of immunity against HAd5, we immunized animals via the i.n. or i.m. route of inoculation with a HAd vector (HAd-HA-NP) expressing the hemagglutinin (HA) and nucleoprotein (NP) of A/Vietnam/1203/04 (H5N1) influenza virus. The immunogenicity and protection results suggest that low levels of vector immunity (<520 virus-neutralization titer) induced by priming mice with up to 10(7) plaque forming units (p.f.u.) of HAd-WT did not adversely impact the protective efficacy of the vaccine. Furthermore, high levels of vector immunity (approximately 1500 virus-neutralization titer) induced by priming mice with 10(8) p.f.u. of HAd-WT were overcome by either increasing the vaccine dose or using alternate routes of vaccination. A further increase in the priming dose to 10(9) p.f.u. allowed only partial protection. These results suggest possible strategies to overcome the variable levels of human immunity against adenoviruses, leading to better utilization of HAd vector-based vaccines.

EphrinA1-EphA2 interaction-mediated apoptosis and FMS-like tyrosine kinase 3 receptor ligand-induced immunotherapy inhibit tumor growth in a breast cancer mouse model.

BACKGROUND: The receptor tyrosine kinase EphA2 is overexpressed in several types of cancers and is currently being pursued as a target for breast cancer therapeutics. The EphA2 ligand EphrinA1 induces EphA2 phosphorylation and intracellular internalization and degradation, thus inhibiting tumor progression. The hematopoietic growth factor, FMS-like tyrosine kinase 3 receptor ligand (Flt3L), promotes expansion and mobilization of functional dendritic cells. METHODS: We tested the EphrinA1-EphA2 interaction in MDA-MB-231 breast cancer cells focusing on the receptor-ligand-mediated apoptosis of breast cancer cells. To determine whether EphrinA1-EphA2 interaction-associated apoptosis and Flt3L-mediated immunotherapy would have an additive effect in inhibiting tumor growth, we used an immunocompetent mouse model of breast cancer to evaluate intratumoral (i.t.) inoculation strategies with human adenovirus (HAd) vectors expressing either EphrinA1 (HAd-EphrinA1-Fc), Flt3L (HAd-Flt3L) or a combination of EphrinA1-Fc + Flt3L (HAd-EphrinA1-Fc + HAd-Flt3L). RESULTS: In vitro analysis demonstrated that an EphrinA1-EphA2 interaction led to apoptosis-related changes in breast cancer cells. In vivo, three i.t. inoculations of HAd-EphrinA1-Fc showed potent inhibition of tumor growth. Furthermore, increased inhibition in tumor growth was observed with the combination of HAd-EphrinA1-Fc and HAd-Flt3L accompanied by the generation of an anti-tumor adaptive immune response. CONCLUSIONS: The results obtained in the present study, indicating the induction of apoptosis and inhibition of mammary tumor growth, show the potential therapeutic benefits of HAd-EphrinA1-Fc. In combination with HAd-Flt3L, this represents a promising strategy for effectively inducing mammary tumor regression by HAd vector-based therapy.

Sequential administration of bovine and human adenovirus vectors to overcome vector immunity in an immunocompetent mouse model of breast cancer.

The potential of a bovine adenovirus serotype 3 (BAd3)-based vector to bypass the human adenoviral serotype 5 (HAd5)-specific neutralizing immune response was evaluated in an immunocompetent mouse model of breast cancer. Initially we monitored vector biodistribution, genome persistence, transgene expression, and potential toxicity of HAd-GFP [HAd5 vector expressing green fluorescent protein (GFP)] or BAd-GFP (BAd3 vector expressing GFP) in FVB/n mice bearing tumors. A comparable biodistribution pattern for BAd-GFP and HAd-GFP was evident. In addition, following the development of vector-specific immune responses, animals were inoculated intratumorally (i.t.) with HAd-GFP or BAd-GFP. HAd-GFP immunity did not hamper the transduction and persistence of BAd-GFP into the tumors and other organs, and, similarly, BAd-GFP immunity did not hamper the transduction and persistence of HAd-GFP. Both BAd3 and HAd5 vectors showed relatively higher transgene expression in the presence of heterologous vector immunity. In contrast, the homologous vector immunity was associated with a rapid vector clearance and decline in transgene expression levels. Histopathological changes in BAd-GFP inoculated animals were generally mild with some acute but recoverable hepatic perturbations. Overall, the data suggest the importance of BAd3 vectors for sequential vector administration in overcoming the vector immunity for cancer gene therapy.

Persistence and the state of bovine and porcine adenoviral vector genomes in human and nonhuman cell lines.

The state of vector genome in transduced cells influences the duration of transgene expression and can be a safety concern if it gets integrated randomly into the host genome. Although human adenovirus (Ad) serotype 5 (HAd5) mainly persists in a linear episomal form, information regarding the state of bovine Ad serotype 3 (BAd3) and porcine Ad serotype 3 (PAd3) vector genomes in human and nonhuman cells is currently unknown. To address this issue, MDA-MB-231 (human), MDBK (bovine), PK-15 (porcine), MT1A2 (mouse) and NIH-3T3 (mouse) cell lines were infected with replication-defective BAd3, PAd3 or HAd5 vectors carrying the green fluorescent protein (GFP) gene. The persistence and the state of vector genome were assessed by quantitative real-time PCR and Southern blot hybridization, respectively. Levels of transgene and Ad gene expressions were quantified using real-time RT-PCR. Persistence of BAd3 or PAd3 vectors was comparable to that of HAd5 vector. Only the linear episomal form of the vector genome was observed with each vector. In addition, expression levels of transgene as well as viral genes by all three vectors were comparable and correlated with their transduction levels in each cell type. These results indicate comparable biologic behavior of BAd3, PAd3 and HAd5 vectors in cell culture.

Production of adenovirus vectors and their use as a delivery system for influenza vaccines.

IMPORTANCE OF THE FIELD: With the emergence of highly pathogenic avian influenza H5N1 viruses that have crossed species barriers and are responsible for lethal infections in humans in many countries, there is an urgent need for the development of effective vaccines which can be produced in large quantities at a short notice and confer broad protection against these H5N1 variants. In order to meet the potential global vaccine demand in a pandemic scenario, new vaccine-production strategies must be explored in addition to the currently used egg-based technology for seasonal influenza. AREAS COVERED IN THIS REVIEW: Adenovirus (Ad) based influenza vaccines represent an attractive alternative/supplement to the currently licensed egg-based influenza vaccines. Ad-based vaccines are relatively inexpensive to manufacture, and their production process does not require either chicken eggs or labor-intensive and time-consuming processes necessitating enhanced biosafety facilities. Most importantly, in a pandemic situation, this vaccine strategy could offer a stockpiling option to reduce the response time before a strain-matched vaccine could be developed. WHAT THE READER WILL GAIN: This review discusses Ad-vector technology and the current progress in the development of Ad-based influenza vaccines. TAKE HOME MESSAGE: Ad vector-based influenza vaccines for pandemic preparedness are under development to meet global vaccine demand.