Replication-competent Vectors Research Articles

Optimizing Pancreatic Cancer Therapy: The Promise of Immune Stimulatory Oncolytic Viruses.

Pancreatic cancer presents formidable challenges due to rapid progression and resistance to conventional treatments. Oncolytic viruses (OVs) selectively infect cancer cells and cause cancer cells to lyse, releasing molecules that can be identified by the host's immune system. Moreover, OV can carry immune-stimulatory payloads such as interleukin-12, which when delivered locally can enhance immune system-mediated tumor killing. OVs are very well tolerated by cancer patients due to their ability to selectively target tumors without affecting surrounding normal tissues. OVs have recently been combined with other therapies, including chemotherapy and immunotherapy, to improve clinical outcomes. Several OVs including adenovirus, herpes simplex viruses (HSVs), vaccinia virus, parvovirus, reovirus, and measles virus have been evaluated in preclinical and clinical settings for the treatment of pancreatic cancer. We evaluated the safety and tolerability of a replication-competent oncolytic adenoviral vector carrying two suicide genes (thymidine kinase, TK; and cytosine deaminase, CD) and human interleukin-12 (hIL12) in metastatic pancreatic cancer patients in a phase 1 trial. This vector was found to be safe and well-tolerated at the highest doses tested without causing any significant adverse events (SAEs). Moreover, long-term follow-up studies indicated an increase in the overall survival (OS) in subjects receiving the highest dose of the OV. Our encouraging long-term survival data provide hope for patients with advanced pancreatic cancer, a disease that has not seen a meaningful increase in OS in the last five decades. In this review article, we highlight several preclinical and clinical studies and discuss future directions for optimizing OV therapy in pancreatic cancer. We envision OV-based gene therapy to be a game changer in the near future with the advent of newer generation OVs that have higher specificity and selectivity combined with personalized treatment plans developed under AI guidance.

Longitudinal Monitoring of the Effects of Anti-Adenoviral Treatment Regimens in a Permissive In Vivo Model.

Adenovirus infections of immunocompromised patients can cause life-threatening disseminated disease. While there are presently no drugs specifically approved to treat these infections, there are several compounds that showed efficacy against adenovirus in preclinical studies. For any such compound, low toxicity is an essential requirement. As cumulative drug effects can accentuate pathology, especially in patients with other morbidities, it is important to limit antiviral exposure to what is absolutely necessary. This is achievable by monitoring the virus burden of the patients and administering antivirals to suppress virus replication to a non-pathogenic level. We modeled such a system using Syrian hamsters infected with a replication-competent adenovirus vector, in which luciferase expression is coupled to virus replication. We found that virus replication could be followed in vivo in the same animal by repeated measurement of luciferase expression. To test the utility of an interrupted treatment regimen, we used NPP-669 and valganciclovir, two antiviral compounds with high and moderate anti-adenoviral efficacy, respectively. We found that short-term treatment of adenovirus-infected hamsters at times of peak virus replication can prevent virus-associated pathology. Thus, we believe that this animal model can be used to model different treatment regimens for anti-adenoviral compounds.

Construction and Characterization of a High-Capacity Replication-Competent Murine Cytomegalovirus Vector for Gene Delivery.

We investigated the basic characteristics of a new murine cytomegalovirus (MCMV) vector platform. Using BAC technology, we engineered replication-competent recombinant MCMVs with deletions of up to 26% of the wild-type genome. To this end, we targeted five gene blocks (m01-m17, m106-m109, m129-m141, m144-m158, and m159-m170). BACs featuring deletions from 18% to 26% of the wild-type genome exhibited delayed virus reconstitution, while smaller deletions (up to 16%) demonstrated reconstitution kinetics similar to those of the wild type. Utilizing an innovative methodology, we introduced large genomic DNA segments, up to 35 kbp, along with reporter genes into a newly designed vector with a potential cloning capacity of 46 kbp (Q4). Surprisingly, the insertion of diverse foreign DNAs alleviated the delayed plaque formation phenotype of Q4, and these large inserts remained stable through serial in vitro passages. With reporter-gene-expressing recombinant MCMVs, we successfully transduced not only mouse cell lines but also non-rodent mammalian cells, including those of human, monkey, bovine, and bat origin. Remarkably, even non-mammalian cell lines derived from chickens exhibited successful transduction.

Update on Managing the Risks of Exposure to Lentiviral and Retroviral Vectors.

This paper aims to review the risks associated with using lentiviral and retroviral vectors in research and clinical settings and to propose an update to an effective treatment plan. Risks of exposure were evaluated based on vector design, safety features, viral tropism, transgene, and means and modes of transmission. These risks were weighed against the potential risks and benefits of current HIV medications. We recommend the following post-exposure prophylactic treatment for significant lentiviral vector exposures: 1) dolutegravir 50 mg. taken once a day for 7 days; and 2) tenofovir disoproxil fumarate 300 mg. taken once a day for 7 days (28 days of both medications for replication-competent vectors). Due to the highly efficient delivery of transgenes by modern lentiviral and retroviral vectors, post-exposure prophylaxis is indicated to prevent vector integration and oncogenic risks.

User-Friendly Replication-Competent MAdV-1 Vector System with a Cloning Capacity of 3.3 Kilobases

Mouse adenoviruses (MAdV) play important roles in studying host–adenovirus interaction. However, easy-to-use reverse genetics systems are still lacking for MAdV. An infectious plasmid pKRMAV1 was constructed by ligating genomic DNA of wild-type MAdV-1 with a PCR product containing a plasmid backbone through Gibson assembly. A fragment was excised from pKRMAV1 by restriction digestion and used to generate intermediate plasmid pKMAV1-ER, which contained E3, fiber, E4, and E1 regions of MAdV-1. CMV promoter-controlled GFP expression cassette was inserted downstream of the pIX gene in pKMAV1-ER and then transferred to pKRMAV1 to generate adenoviral plasmid pKMAV1-IXCG. Replacement of transgene could be conveniently carried out between dual BstZ17I sites in pKMAV1-IXCG by restriction-assembly, and a series of adenoviral plasmids were generated. Recombinant viruses were rescued after transfecting linearized adenoviral plasmids to mouse NIH/3T3 cells. MAdV-1 viruses carrying GFP or firefly luciferase genes were characterized in gene transduction, plaque-forming, and replication in vitro or in vivo by observing the expression of reporter genes. The results indicated that replication-competent vectors presented relevant properties of wild-type MAdV-1 very well. By constructing viruses bearing exogenous fragments with increasing size, it was found that MAdV-1 could tolerate an insertion up to 3.3 kb. Collectively, a replication-competent MAdV-1 vector system was established, which simplified procedures for the change of transgene or modification of E1, fiber, E3, or E4 genes.

A Replication-Competent Retroviral Vector Expressing the HERV-W Envelope Glycoprotein is a Potential Tool for Cancer Gene Therapy.

The fusogenic membrane glycoprotein (FMG) derived from the human endogenous retrovirus-W (HERV-W) exhibits fusogenic properties, making it a promising candidate for cancer gene therapy. When cells are transfected with HERV-W FMG, they can fuse with neighboring cells expressing the receptor, resulting in the formation of syncytia. These syncytia eventually undergo cell death within a few days. In addition, it has been observed that an HERV-W env mutant, which is truncated after amino acid 483, displays increased fusogenicity compared to the wild-type HERV-W env. In this study, we observed syncytium formation upon transfection of HeLa and TE671 human cancer cells with plasmids containing the HERV-W 483 gene. To explore the potential of a semi-replication-competent retroviral (s-RCR) vector encoding HERV-W 483 for FMG-mediated cancer gene therapy, we developed two replication-defective retroviral vectors: a gag-pol vector encoding HERV-W 483 (MoMLV-HERV-W 483) and an env vector encoding VSV-G (pCLXSN-VSV-G-EGFP). When MoMLV-HERV-W 483 and pCLXSN-VSV-G-EGFP were co-transfected into HEK293T cells to produce the s-RCR vector, gradual syncytium formation was observed. However, the titers of the s-RCR virus remained consistently low. To enhance gene transfer efficiency, we constructed an RCR vector encoding HERV-W 483 (MoMLV-10A1-HERV-W 483), which demonstrated replication ability in HEK293T cells. Infection of A549 and HT1080 human cancer cell lines with this RCR vector induced syncytium formation and subsequent cell death. Consequently, both the s-RCR vector and RCR encoding HERV-W 483 hold promise as valuable tools for cancer gene therapy.

Attenuated Replication-Competent Herpes Simplex Virus Expressing an ECM-Modifying Transgene Hyaluronan Synthase 2 of Naked Mole Rat in Oncolytic Gene Therapy.

Herpes simplex virus (HSV) has proven successful in treating human cancer. Since the approval of talimogene laherparepvec (T-VEC) in 2015, HSV has been thoroughly researched to discover novel mechanisms to combat cancer and treat other diseases. Another HSV-based drug, beremagene geperpavec (B-VEC), received approval in 2023 to treat the rare genetic disease dystrophic epidermolysis bullosa, and was also the first clinically approved HSV vector carrying an extracellular matrix (ECM)-modifying transgene. The ECM is a network of macromolecules surrounding cells, which provides support and regulates cell growth and differentiation, the disruption of which is common in cancer. The naked mole rat (NMR) has a thick ECM and a unique mutation in the hyaluronan synthase 2 (HAS2) gene, which has been linked to the high cancer resistance of the species. To study the effect of this mutation in human cancer, we have developed an attenuated, replication-competent HSV vector expressing the NMR-HAS2 gene. The viral replication, transgene expression and cytotoxic effect of the novel vector was studied in glioma cells. Our results show that an attenuated, replication-competent HSV vector expressing a foreign ECM-modifying transgene, namely HAS2, provides an effective tool to study and combat cancer in humans.

Protease-deleted adenovirus as an alternative for replication-competent adenovirus vector

For cancer therapy and vaccination an amplified expression of the therapeutic gene is desired. Previously, we have developed a single-cycle adenovirus vector (SC-AdV) by deleting the adenovirus protease (PS) gene. In order to keep the E1 region intact within the PS-deleted adenoviruses, we examined the insertion of two transgenes under the control of a constitutive or inducible promoters. These were inserted between E4 and the right inverted terminal repeat in a wide variety of backbones with various combinations of PS, E3 and E4 deletion. Our data showed that PS-deleted adenoviruses, expressed transgenes as strongly as replication-competent AdVs in HEK293A and a variant of HeLa cells. In a head-to-head comparison in four human cell lines, we demonstrated that SC-AdV, was comparable for transgene expression efficacy with its replication-competent counterpart. However, the SC-AdV expresses its transgene 10 to 16,000 times higher than its replication-defective counterpart.

Single-dose of a replication-competent adenovirus-vectored vaccine provides sterilizing protection against Rift Valley fever virus challenge

Rift Valley fever virus (RVFV) is one of the most important virulent pathogens causing severe disease in animals and humans. However, there is currently no approved vaccine to prevent RVFV infection in humans. The use of human adenovirus serotype 4 (Ad4) as a vector for an RVFV vaccine has not been reported. Here, we report the generation of a replication-competent recombinant Ad4 vector expressing codon-optimized forms of the RVFV glycoproteins Gn and Gc (named Ad4-GnGc). Intramuscular immunization with Ad4-GnGc elicited robust neutralizing antibodies against RVFV and cellular immune responses in mice. A single low-dose vaccination with Ad4-GnGc completely protected interferon-α/β receptor-deficient A129 mice from lethal RVFV infection. More importantly, Ad4-GnGc efficacy was not affected by pre-existing immunity to adenovirus serotype 5, which currently exists widely in populations. These results suggest that Ad4-GnGc is a promising vaccine candidate against RVFV.

A replication-competent smallpox vaccine LC16m8Δ-based COVID-19 vaccine

ABSTRACT Viral vectors are a potent vaccine platform for inducing humoral and T-cell immune responses. Among the various viral vectors, replication-competent ones are less commonly used for coronavirus disease 2019 (COVID-19) vaccine development compared with replication-deficient ones. Here, we show the availability of a smallpox vaccine LC16m8Δ (m8Δ) as a replication-competent viral vector for a COVID-19 vaccine. M8Δ is a genetically stable variant of the licensed and highly effective Japanese smallpox vaccine LC16m8. Here, we generated two m8Δ recombinants: one harbouring a gene cassette encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein, named m8Δ-SARS2(P7.5-S)-HA; and one encoding the S protein with a highly polybasic motif at the S1/S2 cleavage site, named m8Δ-SARS2(P7.5-SHN)-HA. M8Δ-SARS2(P7.5-S)-HA induced S-specific antibodies in mice that persisted for at least six weeks after a homologous boost immunization. All eight analysed serum samples displayed neutralizing activity against an S-pseudotyped virus at a level similar to that of serum samples from patients with COVID-19, and more than half (5/8) also had neutralizing activity against the Delta/B.1.617.2 variant of concern. Importantly, most serum samples also neutralized the infectious SARS-CoV-2 Wuhan and Delta/B.1.617.2 strains. In contrast, immunization with m8Δ-SARS2(P7.5-SHN)-HA elicited significantly lower antibody titres, and the induced antibodies had less neutralizing activity. Regarding T-cell immunity, both m8Δ recombinants elicited S-specific multifunctional CD8+ and CD4+ T-cell responses even after just a primary immunization. Thus, m8Δ provides an alternative method for developing a novel COVID-19 vaccine.

Residues 140-142, 199-200, 222-223, and 262 in the Surface Glycoprotein of Subgroup A Avian Leukosis Virus Are the Key Sites Determining Tva Receptor Binding Affinity and Infectivity.

Subgroup A avian leukosis virus (ALV-A) invades cells through gp85-encoded surface glycoprotein (SU) via specifically recognizing the cellular receptor Tva. To identify the key residues of ALV-A SU that determine the Tva binding affinity and infectivity in DF-1 cells, a strategy of substituting corresponding residues of SU between ALV-A RSA and ALV-E ev-1 (using Tvb as the receptor) was adopted. A series of chimeric soluble gp85 proteins were expressed for co-immunoprecipitation (co-IP) analysis and blocking analysis of viral entry, and various recombinant viruses based on replication-competent avian retrovirus vectors containing Bryan polymerase (RCASBP) were constructed for transfection into DF-1 cells and measurement of the percentage of GFP-positive cells. The results revealed that the substitution of residues V138, W140, Y141, L142, S145, and L154 of host range region 1 (hr1), residues V199, G200, Q202, R222, and R223 of host range region 2 (hr2), and residue G262 of variable region 3 (vr3) reduced the viral infectivity and Tva binding affinity, which was similar to the effects of the −139S, −151N, −155PWVNPF, −201NFD, Δ214–215, and −266S mutations. Our study indicated that hr1 and hr2 contain the principal receptor interaction determinants, with new identified-vr3 also playing a key role in the receptor binding affinity of ALV-A.

Tumor-targeted fluorescence labeling systems for cancer diagnosis and treatment.

Conventional imaging techniques are available for clinical identification of tumor sites. However, detecting metastatic tumor cells that are spreading from primary tumor sites using conventional imaging techniques remains difficult. In contrast, fluorescence‐based labeling systems are useful tools for detecting tumor cells at the single‐cell level in cancer research. The ability to detect fluorescent‐labeled tumor cells enables investigations of the biodistribution of tumor cells for the diagnosis and treatment of cancer. For example, the presence of fluorescent tumor cells in the peripheral blood of cancer patients is a predictive biomarker for early diagnosis of distant metastasis. The elimination of fluorescent tumor cells without damaging normal tissues is ideal for minimally invasive treatment of cancer. To capture fluorescent tumor cells within normal tissues, however, tumor‐specific activated target molecules are needed. This review focuses on recent advances in tumor‐targeted fluorescence labeling systems, in which indirect reporter labeling using tumor‐specific promoters is applied to fluorescence labeling of tumor cells for the diagnosis and treatment of cancer. Telomerase promoter‐dependent fluorescence labeling using replication‐competent viral vectors produces fluorescent proteins that can be used to detect and eliminate telomerase‐positive tumor cells. Tissue‐specific promoter‐dependent fluorescence labeling enables identification of specific tumor cells. Vimentin promoter‐dependent fluorescence labeling is a useful tool for identifying tumor cells that undergo epithelial–mesenchymal transition (EMT). The evaluation of tumor cells undergoing EMT is important for accurately assessing metastatic potential. Thus, tumor‐targeted fluorescence labeling systems represent novel platforms that enable the capture of tumor cells for the diagnosis and treatment of cancer.

Protease-Deleted Adenovirus as an Alternative for Replication-Competent Adenovirus Vector

Protease-Deleted Adenovirus as an Alternative for Replication-Competent Adenovirus Vector

Susceptibility of Dog, Hamster, and Mouse Cells to the Replication-Competent Adenovirus 11p E1/E3 Green Fluorescence Protein Vector Has Implications for the Selection of Animal Vaccine Models.

Human adenovirus (Ad)-vectored vaccines require viruses that can internalize into host cells and express the vaccine antigen. Evaluation of the expressed antigen in animal cells is a critical step in preclinical trials of viral vaccines. Due to the species specificity of Ads, it is difficult to find a suitable animal model. Thus, in this study, we compared the efficacy of Ad 11 prototype (Ad11p)-mediated green fluorescence protein (GFP) expression in cell lines of dog (MDCK), hamster (CHO), and mouse (McCoy and C127). Although these cell lines did not express the known primary cellular receptors for Ad11p virus infection (i.e., CD46), Ad11pE1GFP could infect and express GFP with various efficacies. For instance, it manifested relatively higher GFP expression in MDCK than in CHO, McCoy, and C127. However, infection leading to efficient viral release was not observed in any of the studied cell lines. The apparent differences were attributed to particularities of mouse and hamster cell lines, which might have led to the repression of viral DNA synthesis and to the low level of GFP expression mediated by Ad11pe3GFP. Moreover, our results revealed that undetectable hexon protein hampered the assembly of virus particles in CHO and MDCK cells. Ad11p differed from Ad5 in the ability for viral DNA synthesis when infecting CHO cells. Although a defective Ad has been successfully developed for SARS-CoV-2 vaccines in clinical applications, it has been difficult to generate one that can be used as an oral SARS-CoV-2 vaccine. Fortunately, our replication-competent Ad 11p vector might solve this problem. Regarding the use of Ad-vector candidates for vaccine purposes, this study demonstrates the selection of animal cell lines and determination of suitable virus doses in in vitro experiments.

Oncolytic Foamy Virus: Generation and Properties of a Nonpathogenic Replicating Retroviral Vector System That Targets Chronically Proliferating Cancer Cells

Nonpathogenic retroviruses of the Spumaretrovirinae subfamily can persist long-term in the cytoplasm of infected cells, completing their lifecycle only after the nuclear membrane dissolves at the time of cell division. Since the targeting of slowly dividing cancer cells remains an unmet need in oncolytic virotherapy we constructed a replication competent Foamy Virus vector (oFV) from the genomes of two chimpanzee Simian Foamy Viruses (PAN1 and PAN2) and inserted a GFP transgene in place of the bel-2 open reading frame. oFV-GFP infected and propagated with slow kinetics in multiple human tumor cell lines, inducing a syncytial cytopathic effect. Infection of growth arrested MRC5 cells was not productive, but oFV genomes persisted in the cytoplasm and the productive viral lifecycle resumed when cell division was later restored. In vivo, the virus propagated extensively in intraperitoneal ovarian cancer xenografts, slowing tumor growth, significantly prolonging survival of the treated mice and sustaining GFP transgene expression for at least 45 days. Our data indicate that oFV is a promising new replication-competent viral and gene delivery platform for efficient targeting of the most fundamental trait of cancer cells, their ability to sustain chronic proliferation.Significance:The infectivity of certain retroviruses is limited to dividing cells, which makes them attractive tools for targeting cancer cell proliferation. Previously developed replication-competent gammaretroviral vectors spread efficiently in rapidly dividing cancer cells, but not in cancer cells that divide more slowly. In contrast to rapidly proliferating transplantable mouse tumors, slow proliferation is a hallmark of human cancers and may have contributed to the clinical failure of the preclinically promising Murine Leukemia Virus vector Toca511 which failed to show efficacy in a phase 3 clinical trial in patients with glioblastoma. The studies presented in our manuscript show that oncolytic Foamy Virus (oFV) vectors are capable of persisting unintegrated in quiescent cells and resuming their life cycle once the cells start dividing again. This property of oFVs, together with their lack of pathogenicity and their ability to catalyze the fusion of infected cancer cells, makes them an attractive platform for further investigation.

Safe and effective two-in-one replicon-and-VLP minispike vaccine for COVID-19: Protection of mice after a single immunization.

Vaccines of outstanding efficiency, safety, and public acceptance are needed to halt the current SARS-CoV-2 pandemic. Concerns include potential side effects caused by the antigen itself and safety of viral DNA and RNA delivery vectors. The large SARS-CoV-2 spike (S) protein is the main target of current COVID-19 vaccine candidates but can induce non-neutralizing antibodies, which might cause vaccination-induced complications or enhancement of COVID-19 disease. Besides, encoding of a functional S in replication-competent virus vector vaccines may result in the emergence of viruses with altered or expanded tropism. Here, we have developed a safe single round rhabdovirus replicon vaccine platform for enhanced presentation of the S receptor-binding domain (RBD). Structure-guided design was employed to build a chimeric minispike comprising the globular RBD linked to a transmembrane stem-anchor sequence derived from rabies virus (RABV) glycoprotein (G). Vesicular stomatitis virus (VSV) and RABV replicons encoding the minispike not only allowed expression of the antigen at the cell surface but also incorporation into the envelope of secreted non-infectious particles, thus combining classic vector-driven antigen expression and particulate virus-like particle (VLP) presentation. A single dose of a prototype replicon vaccine complemented with VSV G, VSVΔG-minispike-eGFP (G), stimulated high titers of SARS-CoV-2 neutralizing antibodies in mice, equivalent to those found in COVID-19 patients, and protected transgenic K18-hACE2 mice from COVID-19-like disease. Homologous boost immunization further enhanced virus neutralizing activity. The results demonstrate that non-spreading rhabdovirus RNA replicons expressing minispike proteins represent effective and safe alternatives to vaccination approaches using replication-competent viruses and/or the entire S antigen.

Efficacy and Safety of Clinical-Grade Human Vascular Endothelial Growth Factor-DΔNΔC Gene Therapy Containing Residual Replication-Competent Adenoviruses.

Biological bypass through induced angiogenesis by vascular endothelial growth factor D (VEGF-D) gene therapy (GT) is a new concept for the treatment of cardiac ischemia. Serotype 5 adenoviruses are used in the clinical trials for transferring the VEGF-D cDNA into the ischemic myocardium. However, the presence of replication-competent vectors in the adenovirus products is a widely recognized problem that may pose a potential safety risk to the treated patients. We compared three different VEGF-D GT production lots containing different levels of replication-competent adenoviruses (RCA) tested in 3 × 1010 viral particles (vp): <10 RCA (VEGF-D L-RCA1), 10-100 RCA (VEGF-D H-RCA2), and 100-200 RCA (VEGF-D H-RCA3), as measured by a novel droplet digital polymerase chain reaction (PCR) RCA assay in a preclinical rabbit model (n = 21). β-galactosidase encoding nonclinical-grade preparation was used as a nonangiogenic control. Each preparation was injected into the right semimembranosus muscle using dose of 1 × 1011 vp. Efficacy of the products was tested by the combination of contrast pulse sequencing ultrasound and modified Miles assay as well as quantifying the total cross-sectional area of capillaries. Safety, immunogenicity, toxicity, biodistribution, and shedding were assessed by general histology, serial measurements of C-reactive protein, white blood cell count and body temperature as well as using quantitative real-time PCR with primers targeted to the VEGF-D and replication-permitting E1 sequences. We found no significant differences in the efficacy or safety between the study groups. Most importantly, no detectable presence of RCA-specific E1 sequence was found in any samples tested, indicating that no detectable vector replication took place in vivo. We conclude that relatively low levels of RCA in adenoviral GT products may not be as important major safety issue as previously anticipated.

Modified Adenovirus Prime-Protein Boost Clade C HIV Vaccine Strategy Results in Reduced Viral DNA in Blood and Tissues Following Tier 2 SHIV Challenge.

Designing immunogens and improving delivery methods eliciting protective immunity is a paramount goal of HIV vaccine development. A comparative vaccine challenge study was performed in rhesus macaques using clade C HIV Envelope (Env) and SIV Gag antigens. One group was vaccinated using co-immunization with DNA Gag and Env expression plasmids cloned from a single timepoint and trimeric Env gp140 glycoprotein from one of these clones (DNA+Protein). The other group was a prime-boost regimen composed of two replicating simian (SAd7) adenovirus-vectored vaccines expressing Gag and one Env clone from the same timepoint as the DNA+Protein group paired with the same Env gp140 trimer (SAd7+Protein). The env genes were isolated from a single pre-peak neutralization timepoint approximately 1 year post infection in CAP257, an individual with a high degree of neutralization breadth. Both DNA+Protein and SAd7+Protein vaccine strategies elicited significant Env-specific T cell responses, lesser Gag-specific responses, and moderate frequencies of Env-specific TFH cells. Both vaccine modalities readily elicited systemic and mucosal Env-specific IgG but not IgA. There was a higher frequency and magnitude of ADCC activity in the SAd7+Protein than the DNA+Protein arm. All macaques developed moderate Tier 1 heterologous neutralizing antibodies, while neutralization of Tier 1B or Tier 2 viruses was sporadic and found primarily in macaques in the SAd7+Protein group. Neither vaccine approach provided significant protection from viral acquisition against repeated titered mucosal challenges with a heterologous Tier 2 clade C SHIV. However, lymphoid and gut tissues collected at necropsy showed that animals in both vaccine groups each had significantly lower copies of viral DNA in individual tissues compared to levels in controls. In the SAd7+Protein-vaccinated macaques, total and peak PBMC viral DNA were significantly lower compared with controls. Taken together, this heterologous Tier 2 SHIV challenge study shows that combination vaccination with SAd7+Protein was superior to combination DNA+Protein in reducing viral seeding in tissues in the absence of protection from infection, thus emphasizing the priming role of replication-competent SAd7 vector. Despite the absence of correlates of protection, because antibody responses were significantly higher in this vaccine group, we hypothesize that vaccine-elicited antibodies contribute to limiting tissue viral seeding.

Single Amino Acids G196 and R198 in hr1 of Subgroup K Avian Leukosis Virus Glycoprotein Are Critical for Tva Receptor Binding.

Avian leukosis viruses (ALVs), a type of retrovirus responsible for various tumor diseases in chickens, are divided into 11 subgroups: ALV-A to ALV-K. After the envelope glycoproteins of ALV interact with the cellular receptor to initiate viral invasion, alterations in a few amino acids of the viral glycoproteins or cell receptors may trigger changes in their conformation and binding affinity. To identify the functional determinants of the ALV-K envelope protein that binds to Tva (a recently identified cellular receptor of ALV-K), using the strategy of continuous, segment-by-segment substitution of the gp85-encoded surface glycoprotein (SU) of ALV-K GDFX0602 with ALV-E ev-1 (using Tvb as the receptor), a series of chimeric soluble gp85 proteins were expressed for co-immunoprecipitation (co-IP) analysis and a series of recombinant viruses with replication-competent avian retrovirus vectors containing Bryan polymerase (RCASBP) as their skeleton were created for transfecting to DF-1 cells and titer determination. The co-IP analysis, fluorescence-activated cell sorting, and virus titer measurements revealed that the substitution of residues 194–198, 206–216 of hr1, residues 251–256 between hr1 and hr2, and residues 269–280 of hr2 were identified to reduce the binding of gp85 to Tva. The substitution of residues 194–221 in hr1 nullified the infectiveness of these viruses, similar to the effect of single amino acid mutations in K251E and L252I located between hr1 and hr2; continuous amino acid mutations in hr2 could not produce the same effect despite reducing their infectiveness. Finally, single amino acid mutations G196A and R198H nearly abolished the binding of gp85 to Tva and nullified the infectiveness of these viruses to DF-1. This study paves the way for exploring the molecular mechanisms of the binding of Tva to ALV-K SU.

Characterization of rVSVΔG-ZEBOV-GP glycoproteins using automated capillary western blotting

Ebolavirus (EBOV) entry to host cells requires membrane-associated glycoprotein (GP). A recombinant vesicular stomatitis virus vector carrying Zaire Ebola virus glycoprotein (rVSV-ZEBOV) was developed as a vaccine against ebolaviruses. The VSV glycoprotein gene was deleted (rVSVΔG) and ZEBOV glycoprotein (GP) was inserted into the deleted VSV glycoprotein open reading frame (ORF) resulting in a live, replication-competent vector (rVSVΔG-ZEBOV-GP). Automated capillary westerns were used to characterize the rVSVΔG-ZEBOV-GP vaccine (ERVEBO®) manufacturing process with regards to glycoprotein (GP) structure and variants. The method shows a unique electropherogram profile for each process step which could be used to monitor process robustness. rVSVΔG-ZEBOV-GP encodes GP (GP1-GP2), secreted GP (sGP), and small secreted GP (ssGP) variants. Furthermore, a TACE-like activity was observed indirectly by detecting soluble GP2Δ after virus precipitation by ultracentrifugation. Capillary western blotting techniques can guide process development by evaluating process steps such as enzyme treatment. In addition, the technique can assess GP stability and process lot-to-lot consistency. Finally, capillary western-based technology was used to identify a unique biochemical profile of the rVSVΔG-ZEBOV-GP vaccine strain in final product. Virion membrane-bound GP1-GP2 is critical to vaccine-elicited protection by providing both neutralizing antibodies and T-cell response.