The Biodistribution of the Spike Protein after Ad26.COV2.S Vaccination Is Unlikely to Play a Role in Vaccine-Induced Immune Thrombotic Thrombocytopenia.

Ad26.COV2.S vaccination can lead to vaccine-induced immune thrombotic thrombocytopenia (VITT), a rare but severe adverse effect, characterized by thrombocytopenia and thrombosis. The mechanism of VITT induction is unclear and likely multifactorial, potentially including the activation of platelets and endothelial cells mediated by the vaccine-encoded spike protein (S protein). Here, we investigated the biodistribution of the S protein after Ad26.COV2.S dosing in three animal models and in human serum samples. The S protein was transiently present in draining lymph nodes of rabbits after Ad26.COV2.S dosing. The S protein was detected in the serum in all species from 1 day to 21 days after vaccination with Ad26.COV2.S, but it was not detected in platelets, the endothelium lining the blood vessels, or other organs. The S protein S1 and S2 subunits were detected at different ratios and magnitudes after Ad26.COV2.S or COVID-19 mRNA vaccine immunization. However, the S1/S2 ratio did not depend on the Ad26 platform, but on mutation of the furin cleavage site, suggesting that the S1/S2 ratio is not VITT related. Overall, our data suggest that the S-protein biodistribution and kinetics after Ad26.COV2.S dosing are likely not main contributors to the development of VITT, but other S-protein-specific parameters require further investigation.

Peak transgene expression after intramuscular immunization of mice with adenovirus 26-based vector vaccines correlates with transgene-specific adaptive immune responses.

Non-replicating adenovirus-based vectors have been broadly used for the development of prophylactic vaccines in humans and are licensed for COVID-19 and Ebola virus disease prevention. Adenovirus-based vectored vaccines encode for one or more disease specific transgenes with the aim to induce protective immunity against the target disease. The magnitude and duration of transgene expression of adenovirus 5- based vectors (human type C) in the host are key factors influencing antigen presentation and adaptive immune responses. Here we characterize the magnitude, duration, and organ biodistribution of transgene expression after single intramuscular administration of adenovirus 26-based vector vaccines in mice and evaluate the differences with adenovirus 5-based vector vaccine to understand if this is universally applicable across serotypes. We demonstrate a correlation between peak transgene expression early after adenovirus 26-based vaccination and transgene-specific cellular and humoral immune responses for a model antigen and SARS-CoV-2 spike protein, independent of innate immune activation. Notably, the memory immune response was similar in mice immunized with adenovirus 26-based vaccine and adenovirus 5-based vaccine, despite the latter inducing a higher peak of transgene expression early after immunization and a longer duration of transgene expression. Together these results provide further insights into the mode of action of adenovirus 26-based vector vaccines.

Intravenous Administration of Ad26.COV2.S Does Not Induce Thrombocytopenia or Thrombotic Events or Affect SARS-CoV-2 Spike Protein Bioavailability in Blood Compared with Intramuscular Vaccination in Rabbits.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a very rare but serious adverse reaction that can occur after Ad26.COV2.S vaccination in humans, leading to thrombosis at unusual anatomic sites. One hypothesis is that accidental intravenous (IV) administration of Ad26.COV2.S or drainage of the vaccine from the muscle into the circulatory system may result in interaction of the vaccine with blood factors associated with platelet activation, leading to VITT. Here, we demonstrate that, similar to intramuscular (IM) administration of Ad26.COV2.S in rabbits, IV dosing was well tolerated, with no significant differences between dosing routes for the assessed hematologic, coagulation time, innate immune, or clinical chemistry parameters and no histopathologic indication of thrombotic events. For both routes, all other non-adverse findings observed were consistent with a normal vaccine response and comparable to those observed for unrelated or other Ad26-based control vaccines. However, Ad26.COV2.S induced significantly higher levels of C-reactive protein on day 1 after IM vaccination compared with an Ad26-based control vaccine encoding a different transgene, suggesting an inflammatory effect of the vaccine-encoded spike protein. Although based on a limited number of animals, these data indicate that an accidental IV injection of Ad26.COV2.S may not represent an increased risk for VITT.

Cell entry and innate sensing shape adaptive immune responses to adenovirus-based vaccines.

Nonreplicating adenovirus-based vectors have been successfully implemented as prophylactic vaccines against infectious viral diseases and induce protective cellular and humoral responses. Differences in the mechanisms of cellular entry or endosomal escape of these vectors contribute to differences in innate immune sensing between adenovirus species. Innate immune responses to adenovirus-based vaccines, such as interferon signaling, have been reported to affect the development of adaptive responses in preclinical studies, although limited data are available in humans. Understanding the mechanisms of these early events is critical for the development of vaccines that elicit effective and durable adaptive immune responses while maintaining an acceptable reactogenicity profile.

Sequential use of Ad26-based vaccine regimens in NHP to induce immunity against different disease targets.

The adenovirus (Ad)26 serotype-based vector vaccine Ad26.COV2.S has been used in millions of subjects for the prevention of COVID-19, but potentially elicits persistent anti-vector immunity. We investigated if vaccine-elicited immunity to Ad26 vector-based vaccines significantly influences antigen-specific immune responses induced by a subsequent vaccination with Ad26 vector-based vaccine regimens against different disease targets in non-human primates. A homologous Ad26 vector-based vaccination regimen or heterologous regimens (Ad26/Ad35 or Ad26/Modified Vaccinia Ankara [MVA]) induced target pathogen-specific immunity in animals, but also persistent neutralizing antibodies and T-cell responses against the vectors. However, subsequent vaccination (interval, 26-57 weeks) with homologous and heterologous Ad26 vector-based vaccine regimens encoding different target pathogen immunogens did not reveal consistent differences in humoral or cellular immune responses against the target pathogen, as compared to responses in naïve animals. These results support the sequential use of Ad26 vector-based vaccine regimens targeting different diseases.

Human AdV-20-42-42, a Promising Novel Adenoviral Vector for Gene Therapy and Vaccine Product Development.

Preexisting immune responses toward adenoviral vectors limit the use of a vector based on particular serotypes and its clinical applicability for gene therapy and/or vaccination. Therefore, there is a significant interest in vectorizing novel adenoviral types that have low seroprevalence in the human population. Here, we describe the discovery and vectorization of a chimeric human adenovirus, which we call HAdV-20-42-42. Full-genome sequencing revealed that this virus is closely related to human serotype 42, except for the penton base, which is derived from serotype 20. The HAdV-20-42-42 vector could be propagated stably to high titers on existing E1-complementing packaging cell lines. Receptor-binding studies revealed that the vector utilized both CAR and CD46 as receptors for cell entry. Furthermore, the HAdV-20-42-42 vector was potent in transducing human and murine cardiovascular cells and tissues, irrespective of the presence of blood coagulation factor X. In vivo characterizations demonstrate that when delivered intravenously (i.v.) in mice, HAdV-20-42-42 mainly targeted the lungs, liver, and spleen and triggered robust inflammatory immune responses. Finally, we demonstrate that potent T-cell responses against vector-delivered antigens could be induced upon intramuscular vaccination in mice. In summary, from the data obtained we conclude that HAdV-20-42-42 provides a valuable addition to the portfolio of adenoviral vectors available to develop efficacious products in the fields of gene therapy and vaccination. IMPORTANCE Adenoviral vectors are under investigation for a broad range of therapeutic indications in diverse fields, such as oncology and gene therapy, as well as for vaccination both for human and veterinary use. A wealth of data shows that preexisting immune responses may limit the use of a vector. Particularly in the current climate of global pandemic, there is a need to expand the toolbox with novel adenoviral vectors for vaccine development. Our data demonstrate that we have successfully vectorized a novel adenovirus type candidate with low seroprevalence. The cell transduction data and antigen-specific immune responses induced in vivo demonstrate that this vector is highly promising for the development of gene therapy and vaccine products.

Incorporating SPACES recommendations to the COVID-19 ward care approach at the Royal Bournemouth Hospital.

The British Thoracic Society have published SPACES (Sharing Patient Assessments Cuts Exposure for Staff) guidance recommending the use of telecommunications as a means of inpatient clinical assessment during the COVID-19 pandemic. The aim of this audit was to reduce face-to-face exposure time during inpatient care of patients with suspected/confirmed COVID-19 using a telecommunications-based approach. 76 patients were included in the audit. 46 patients were included in cycle 1, which measured the average face-to-face time for clinical consultation per patient. 30 patients were included in cycle 2, whereby history-taking was conductedusing telecommunications and, if required, face-to-face physical examination.Average face-to-face exposure time was reduced to a median of 0 seconds (IQR 0-146.3 seconds) in the telecommunications group, from a median of 312.5 seconds (IQR 178.8-442.3 seconds) in the comparator group (p<0.0001). Patient satisfaction was high with individuals' responses revealing a perception of improved safety by maintaining social distancing. No adverse events were noted.The audit confirms telecommunications can be integrated successfully into the daily inpatient ward round structure, reducing face-to-face exposure time while maintaining patient satisfaction and safety.

Dual Synthetic Peptide Conjugate Vaccine Simultaneously Triggers TLR2 and NOD2 and Activates Human Dendritic Cells.

Simultaneous triggering of Toll-like receptors (TLRs) and NOD-like receptors (NLRs) has previously been shown to synergistically activate monocytes, dendritic cells, and macrophages. We applied these properties in a T-cell vaccine setting by conjugating the NOD2-ligand muramyl-dipeptide (MDP) and TLR2-ligand Pam3CSK4 to a synthetic peptide derived from a model antigen. Stimulation of human DCs with the MDP-peptide-Pam3CSK4 conjugate led to a strongly increased secretion of pro-inflammatory and Th1-type cytokines and chemokines. We further show that the conjugated ligands retain their ability to trigger their respective receptors, while even improving NOD2-triggering. Also, activation of murine DCs was enhanced by the dual triggering, ultimately leading to effective induction of vaccine-specific T cells expressing IFNγ, IL-2, and TNFα. Together, these data indicate that the dual MDP-SLP-Pam3CSK4 conjugate constitutes a chemically well-defined vaccine approach that holds promise for the use in the treatment of virus infections and cancer.

The Nd:YAG laser as first-line treatment for fibrin pupillary-block glaucoma following uncomplicated cataract surgery.

A 43-year-old lady presented with painful, loss of vision in the right eye 2 weeks after cataract surgery. This is on a background of previous central retinal vein occlusion in the right eye and bilateral diabetic retinopathy. Her past medical history included mixed connective tissue disease for which she is on systemic immunosuppressants. Ocular examination revealed significant corneal oedema, a shallow anterior chamber with an intraocular pressure of 45 mmHg. Intraocular pressure lowering medications were administered and repeat examination identified a fine fibrinous membrane attached to the pupillary margin with a substantial third space that had formed between the posterior edge of the membrane and the anterior lens surface. The fibrin membrane was removed with Nd:YAG laser and intraocular pressure reduced to 12 mmHg. Post-procedural management included hourly dexamethasone 0.1% eye drops for 1 week and cyclopentolate 1%. There was no recurrence in the fibrin membrane.

Novel TLR2-binding adjuvant induces enhanced T cell responses and tumor eradication.

BACKGROUND: Ligands for the Toll-like receptor (TLR) family can induce activation of cells of the innate immune system and are widely studied for their potential to enhance adaptive immunity. Conjugation of TLR2-ligand Pam3CSK4 to synthetic long peptides (SLPs) was shown to strongly enhance the induction of antitumor immunity. To further improve cancer vaccination, we have previously shown that the novel TLR2-L Amplivant (AV), a modified Pam3CSK4, potentiates the maturation effects on murine DCs. In the current study, we further assessed the immunological properties of AV. METHODS: Naïve mice were vaccinated with a conjugate of either Pam3CSK4 or AV and an SLP to assess specific T cell priming efficiency in vivo. The potency of AV and Pam3CSK4, either as free compounds or conjugated to different SLPs, to mature murine DCs was compared by stimulating murine dendritic cells overnight followed by ELISA and flow cytometry analysis. Murine tumor experiments were carried out by vaccinating mice carrying established HPV16 E6 and E7-expressing tumors and subsequently analyzing myeloid and lymphoid cells infiltrating the tumor microenvironment. Furthermore, tumor outgrowth after vaccination was monitored to enable comparison of the efficiency to induce antitumor immunity by Pam3CSK-SLP and AV-SLP conjugates. To enhance therapeutic efficacy, AV-SLP conjugate vaccination was combined with ablative therapies to assess whether synergism between such therapies would occur. RESULTS: SLPs conjugated to AV induce stronger DC maturation, in vivo T cell priming and antitumor immunity compared to conjugates with Pam3CSK4. Interestingly, AV-SLP conjugates modulate the macrophage populations in the tumor microenvironment, correlating with a therapeutic effect in an aggressive murine tumor model. The potency of AV-SLP conjugates in cancer vaccination operates optimally in combination with chemotherapy or photodynamic therapy. CONCLUSION: These data allow further optimization of vaccination-based immunotherapy of cancer by use of the improved TLR2-ligand Amplivant.

Adenovirus based HPV L2 vaccine induces broad cross-reactive humoral immune responses.

Oncogenic high-risk human papillomavirus (HPV) infections cause a substantial number of genital and non-genital cancers worldwide. Approximately 70% of all cervical cancers are caused by the high-risk HPV16 and 18 types. The remaining 30% can be attributed to twelve other high-risk HPV-types. Highly efficacious 2-valent, 4-valent and 9-valent L1 protein based prophylactic HPV vaccines are available however with limited cross-protection. To further increase the coverage, development of a multivalent cross-protective HPV vaccine is currently focused on the conserved N-terminus of HPV's L2 protein. We have developed a vaccine candidate based on the rare human adenovirus type 35 (HAdV35) vector that displays a concatemer of L2 protein epitopes from four different HPV-types via protein IX (pIX). A mix of two heterologous HAdV35 pIX-L2 display vectors present highly conserved linear epitopes of nine HPV-types. Each HAdV35 pIX-L2 display vector exhibits a good manufacturability profile. HAdV35 pIX-L2 display vaccine vectors were immunogenic and induced neutralizing antibodies against HPV-types included in the vaccine and cross-neutralizing antibodies against distant a HPV-type not included in the vaccine in mice. The HAdV35 pIX-L2 display vectors offer an opportunity for a multivalent HAdV-based prophylactic HPV vaccine.

A Rare Presentation of Actinic Keratosis Affecting the Tarsal Conjunctiva and Review of the Literature.

We report an unusual case of actinic keratosis (AK) of the tarsal conjunctiva in a 63-year-old man. Examination revealed a crusty, leukoplakic lesion prone to bleeding on the tarsal conjunctiva of the right upper eyelid. This was treated by surgical excisional biopsy. At 1-year follow-up, there was no evidence of recurrence and the surgical site was completely healed without conjunctival scarring. Current opinion cites excessive exposure to ultraviolet (UV) radiation, in particular UV-B in sunlight, as the causative agent in developing AK. In the case we present, the tarsal conjunctiva is an unusual place for actinic keratosis due to the lack of direct UV-light exposure. The key learning points are to evert the upper eyelid during examination especially if the lesion involves the eyelid margin and, secondly, to ensure risk factors are addressed during the history.

Adenovirus vector-based prime-boost vaccination via heterologous routes induces cervicovaginal CD8+ T cell responses against HPV16 oncoproteins.

Recent advances in immunotherapy against cancer underscore the importance of T lymphocytes and tumor microenvironment, but few vaccines targeting cancer have been approved likely due in part to the dearth of common tumor antigens, insufficient immunogenicity and the evolution of immune evasion mechanisms during the progression to malignancy. Human papillomaviruses (HPVs) are the primary etiologic agents of cervical cancer and progression from persistent HPV-infection to cervical intraepithelial lesions and eventually cancer requires persistent expression of the oncoproteins E6 and E7. This offers the opportunity to specifically target these virus-specific antigens for vaccine-induced clearance of infected cells before cancers develop. Here we have evaluated the immunogenicity of Adenovirus Types 26 and 35 derived vectors expressing a fusion of HPV16 E6 and E7 oncoproteins after intramuscular (IM) and/or intravaginal (Ivag) immunization in mice. The adenovirus vectors were shown to transduce an intact cervicovaginal epithelium. IM prime followed by Ivag boost maximized the induction and trafficking of HPV-specific CD8+ T cells producing IFN-γ and TNF-α to the cervicovaginal tract. Importantly, the cervicovaginal CD8+ T cells expressed CD69 and CD103; hallmarks of intraepithelial tissue-resident memory CD8+ T cells. This prime-boost strategy targeting heterologous locations also induced circulating HPV-specific CD8+ T cell responses. Our study prompts further evaluation of Ivag immunization with adenoviral vectors expressing modified E6 and E7 antigens for therapeutic vaccination against persistent HPV infection and cervical intraepithelial neoplasia.

Generation and characterization of a novel candidate gene therapy and vaccination vector based on human species D adenovirus type 56.

The vectorization of rare human adenovirus (HAdV) types will widen our knowledge of this family and their interaction with cells, tissues and organs. In this study we focus on HAdV-56, a member of human Ad species D, and create ease-of-use cloning systems to generate recombinant HAdV-56 vectors carrying foreign genes. We present in vitro transduction profiles for HAdV-56 in direct comparison to the most commonly used HAdV-5-based vector. In vivo characterizations demonstrate that when it is delivered intravenously (i.v.) HAdV-56 mainly targets the spleen and, to a lesser extent, the lungs, whilst largely bypassing liver transduction in mice. HAdV-56 triggered robust inflammatory and cellular immune responses, with higher induction of IFNγ, TNFα, IL5, IL6, IP10, MCP1 and MIG1 compared to HAdV-5 following i.v. administration. We also investigated its potential as a vaccine vector candidate by performing prime immunizations in mice with HAdV-56 encoding luciferase (HAdV-56-Luc). Direct comparisons were made to HAdV-26, a highly potent human vaccine vector currently in phase II clinical trials. HAdV-56-Luc induced luciferase 'antigen'-specific IFNγ-producing cells and anti-HAdV-56 neutralizing antibodies in Balb/c mice, demonstrating a near identical profile to that of HAdV-26. Taken together, the data presented provides further insight into human Ad receptor/co-receptor usage, and the first report on HAdV-56 vectors and their potential for gene therapy and vaccine applications.

Intravitreal injections service: a patient experience evaluation.

INTRODUCTION: This survey has been conducted following the introduction of nurse-led intravitreal injections clinics at the Great Western Hospital. A team of 5 nurses regularly carry out an average of 90 injections per week. METHODS: A series of 169 consecutive injection patients have been offered the study questionnaire between 28 January 2016 and 28 February 2016; patients with no previous experience with a nurse injector were excluded. RESULTS: 76.9% strongly agree that the nurse is more friendly and sensitive to their needs. Only 12.4% strongly agree that a doctor will be more suitable to deliver the injections. Seventy-five percent stated that they were very happy with the care, while 0.4% thought a nurse would be unsafe; 69.2% feel more comfortable asking questions when with a nurse injector. Anxiety and nervousness were found to be the main two symptoms experienced by patients around the time of injections (27.8% and 34.9%, respectively). DISCUSSION: The questionnaire was designed based on the feedback from the local friends and family test. A clear majority of patients gave highly positive feedback about nurse injectors. Considering patients' individual needs at the time of injections, such as allergies or points that needs clarification, helps in overcoming some of the psychological complications of treatment.

Development of a replication-deficient adenoviral vector-based vaccine candidate for the interception of HPV16- and HPV18-induced infections and disease.

High-risk Human papilloma virus (HPV) types are the causative agents of cervical cancer and several other anogenital malignancies. The viral proteins expressed in the (pre)malignant cells are considered ideal targets for immunological intervention. Many approaches have been evaluated for this purpose, mostly aiming at the induction of HPV16 E7- and/or E6-specific cellular immunogenicity. As clinical success has so far been limited, novel approaches are required. We describe the development and pre-clinical testing of a vaccine candidate consisting of replication-deficient adenovirus type 26 and 35 based vectors for the interception of HPV16- and HPV18-related disease. We developed HPV16- and HPV18-specific antigens consisting of fusion proteins of E2, E6 and E7. The vaccine will be suitable for every disease stage, from incident and persistent infections where E2 is predominantly expressed up to late stages where E6 and E7 expression are upregulated. Importantly E6 and E7 are present as reordered fragments to abrogate the transforming activity of these two proteins. Loss of transforming activity was demonstrated in different in vitro models. Robust T-cell immunogenicity was induced upon immunization of mice with the vaccine candidate. Finally, the developed vaccine vectors showed considerable therapeutic efficacy in the TC-1 mouse model. The absence of transforming activity of the antigens and the favorable immunogenicity profile of the adenovirus based vectors along with the fact that these vectors can be readily produced on a large scale makes this approach attractive for clinical evaluation.