Transgene expression knock-down in recombinant Modified Vaccinia virus Ankara vectors improves genetic stability and sustained transgene maintenance across multiple passages.

Modified vaccinia virus Ankara is a versatile vaccine vector, well suited for transgene delivery, with an excellent safety profile. However, certain transgenes render recombinant MVA (rMVA) genetically unstable, leading to the accumulation of mutated rMVA with impaired transgene expression. This represents a major challenge for upscaling and manufacturing of rMVA vaccines. To prevent transgene-mediated negative selection, the continuous avian cell line AGE1.CR pIX (CR pIX) was modified to suppress transgene expression during rMVA generation and amplification. This was achieved by constitutively expressing a tetracycline repressor (TetR) together with a rat-derived shRNA in engineered CR pIX PRO suppressor cells targeting an operator element (tetO) and 3' untranslated sequence motif on a chimeric poxviral promoter and the transgene mRNA, respectively. This cell line was instrumental in generating two rMVA (isolate CR19) expressing a Macaca fascicularis papillomavirus type 3 (MfPV3) E1E2E6E7 artificially-fused polyprotein following recombination-mediated integration of the coding sequences into the DelIII (CR19 M-DelIII) or TK locus (CR19 M-TK), respectively. Characterization of rMVA on parental CR pIX or engineered CR pIX PRO suppressor cells revealed enhanced replication kinetics, higher virus titers and a focus morphology equaling wild-type MVA, when transgene expression was suppressed. Serially passaging both rMVA ten times on parental CR pIX cells and tracking E1E2E6E7 expression by flow cytometry revealed a rapid loss of transgene product after only few passages. PCR analysis and next-generation sequencing demonstrated that rMVA accumulated mutations within the E1E2E6E7 open reading frame (CR19 M-TK) or deletions of the whole transgene cassette (CR19 M-DelIII). In contrast, CR pIX PRO suppressor cells preserved robust transgene expression for up to 10 passages, however, rMVAs were more stable when E1E2E6E7 was integrated into the TK as compared to the DelIII locus. In conclusion, sustained knock-down of transgene expression in CR pIX PRO suppressor cells facilitates the generation, propagation and large-scale manufacturing of rMVA with transgenes hampering viral replication.

Preferential Expansion of HPV16 E1-Specific T Cells from Healthy Donors' PBMCs after Ex Vivo Immunization with an E1E2E6E7 Fusion Antigen.

Persistent human papillomavirus (HPV) infection is responsible for practically all cervical and a high proportion of anogenital and oropharyngeal cancers. Therapeutic HPV vaccines in clinical development show great promise in improving outcomes for patients who mount an anti-HPV T-cell response; however, far from all patients elicit a sufficient immunological response. This demonstrates a translational gap between animal models and human patients. Here, we investigated the potential of a new assay consisting of co-culturing vaccine-transduced dendritic cells (DCs) with syngeneic, healthy, human peripheral blood mononuclear cells (PBMCs) to mimic a human in vivo immunization. This new promising human ex vivo PBMC assay was evaluated using an innovative therapeutic adenovirus (Adv)-based HPV vaccine encoding the E1, E2, E6, and E7 HPV16 genes. This new method allowed us to show that vaccine-transduced DCs yielded functional effector T cells and unveiled information on immunohierarchy, showing E1-specific T-cell immunodominance over time. We suggest that this assay can be a valuable translational tool to complement the known animal models, not only for HPV therapeutic vaccines, and supports the use of E1 as an immunotherapeutic target. Nevertheless, the findings reported here need to be validated in a larger number of donors and preferably in patient samples.

The Insertion of an Evolutionary Lost Four-Amino-Acid Cytoplasmic Tail Peptide into a Syncytin-1 Vaccine Increases T- and B-Cell Responses in Mice.

Human endogenous retrovirus type W (HERV-W) is expressed in various cancers. We previously developed an adenovirus-vectored cancer vaccine targeting HERV-W by encoding an assembled HERV-W group-specific antigen sequence and the HERV-W envelope sequence Syncytin-1. Syncytin-1 is constitutively fusogenic and forms large multinucleated cell fusions when overexpressed. Consequently, immunising humans with a vaccine encoding Syncytin-1 can lead to the formation of extensive syncytia, which is undesirable and poses a potential safety issue. Here, we show experiments in cell lines that restoring an evolutionary lost cleavage site of the fusion inhibitory R-peptide of Syncytin-1 inhibit cell fusion. Interestingly, this modification of the HERV-W vaccine's fusogenicity increased the expression of the vaccine antigens in vitro. It also enhanced Syncytin-1-specific antibody responses and CD8+-mediated T-cell responses compared to the wildtype vaccine in vaccinated mice, with a notable enhancement in responses to subdominant T-cell epitopes but equal responses to dominant epitopes and similar rates of survival following a tumour challenge. The impairment of cell-cell fusion and the enhanced immunogenicity profile of this HERV-W vaccine strengthens the prospects of obtaining a meaningful immune response against HERV-W in patients with HERV-W-overexpressing cancers.

Transforaminal endoscopic thoracic discectomy: surgical technique.

The major challenge inherent to the surgical treatment of thoracic disc herniations is that the disc herniation is often ventral to the spinal cord. Posterior approaches are difficult and dangerous due to the morbidity associated with retraction of the thoracic spinal cord. A ventral approach is not feasible due to the thoracic viscera. A lateral transcavitary approach is the standard for treating ventral thoracic disc pathology but is also quite morbid. Transforaminal endoscopic spine surgery has emerged as a minimally invasive technique for treating thoracic disc pathology and it can be performed in the outpatient setting even with the patient awake. Advances in endoscopic camera technologies as well as the availability of specialty instruments that can be used down a working channel endoscope has now made a myriad of spine pathologies accessible to the minimally invasive spine surgeon. The transforaminal approach and the angled endoscopic camera are an ideal combination for creating a technical advantage to accessing thoracic disc pathology in a minimally invasive fashion. The principal challenges to the approach are needle targeting and understanding the endoscopic visual anatomy. Many surgeons interested in pursuing this technique are often deterred by the burden of the cost and time it takes to become adept and performing the technique. Detailed here are the authors' step-by-step technique and illustrative video that demonstrate transforaminal endoscopic thoracic discectomy (TETD).

Human Ad19a/64 HERV-W Vaccines Uncover Immunosuppression Domain-Dependent T-Cell Response Differences in Inbred Mice.

Expression of human endogenous retrovirus type W (HERV-W) has been linked to cancer, making HERV-W antigens potential targets for therapeutic cancer vaccines. In a previous study, we effectively treated established tumours in mice by using adenoviral-vectored vaccines targeting the murine endogenous retrovirus envelope and group-specific antigen (Gag) of melanoma-associated retrovirus (MelARV) in combination with anti-PD-1. To break the immunological tolerance to MelARV, we mutated the immunosuppressive domain (ISD) of the MelARV envelope. However, reports on the immunogenicity of the HERV-W envelope, Syncytin-1, and its ISD are conflicting. To identify the most effective HERV-W cancer vaccine candidate, we evaluated the immunogenicity of vaccines encoding either the wild-type or mutated HERV-W envelope ISD in vitro and in vivo. Here, we show that the wild-type HERV-W vaccine generated higher activation of murine antigen-presenting cells and higher specific T-cell responses than the ISD-mutated counterpart. We also found that the wild-type HERV-W vaccine was sufficient to increase the probability of survival in mice subjected to HERV-W envelope-expressing tumours compared to a control vaccine. These findings provide the foundation for developing a therapeutic cancer vaccine targeting HERV-W-positive cancers in humans.

Consumers' body image expressions: Reflection of a Snow White or an Evil Queen.

Introduction: The aim of this paper is to explore how minimal-self impacts the body image, projecting it as a reflection of one's approach toward their health and mental well-being. Methods: The study takes qualitative data from two countries India and Germany and draws on a qualitative study of 20 individuals who are involved in some kind of physical activity for a long time. This paper examines the body image perspectives from Grimms Brothers fairytale characters showcasing fit and healthy perspectives on Snow White side and projected and superfluous perspectives on Evil Queen side. The study also provides a model deciphering the rationale for both the reflections. Results: The body image projection from Snow White perspectives (success & dedication, self-esteem, bodybuilding, and cosmetic surgery) relates to positive reflection of oneself with focus on fitness, discipline, and mental rejuvenation in life. Notably, Evil Queen perspectives (unrealistic makeover, dark side of social media, gain an edge over others, and mental benchmarking with fair skin) reveal these facets as motivators to equip their body as means of physical non-verbal communication assets. Conclusion: Analysis shows that there is no clear white or black view of health and fitness projection via body image but it's a gray line that gives wholesome fitness either a holistic mental peace or a competitive or success-oriented approach.

Surgeon reported practice patterns related to full endoscopic cervical decompression procedures.

BACKGROUND: The microsurgical anterior approach to the cervical spine is commonplace. Fewer surgeons perform posterior cervical microsurgical procedures on a routine basis for lack of indication, more bleeding, persistent postoperative neck pain, and risk of progressive misalignment. In comparison, the endoscopic technique is preferentially performed through the posterior approach. Many spine surgeons and even surgeons versed in lumbar endoscopy are often reluctant to consider endoscopic procedures in the cervical spine. We report the results of a surgeon survey to find out why. METHODS: A questionnaire of 10 questions was sent to spine surgeons by email and chat groups in social media networks including Facebook, WeChat, WhatsApp, and LinkedIn to collect practice pattern data about microscopic and endoscopic spine surgery in the lumbar and cervical spine. The responses were cross-tabulated by surgeons' demographic data. Pearson Chi-Square measures, Kappa statistics, and linear regression analysis of agreement or disagreement were performed by analyzing the distribution of variances using the statistical package SPSS Version 27.0. RESULTS: The survey response rate was 39.7%, with 50 of the 126 surgeons who started the survey submitting a completed questionnaire. Of the 50 surgeons, 56.2% were orthopedic, and 42% neurological surgeons. Most surgeons worked in private practice (42%). Another 26% were university-employed, 18% were in private practice affiliated with a university, and the remaining 14% were hospital employed. The majority of surgeons (55.1%) were autodidacts. The largest responding surgeon groups were between 35-44 years (38%) and between 45-54 years of age (34%). Half of the responding surgeons were routinely performing endoscopic cervical spine surgery. The other half did not perform it for the main hurdle of fear of complications (50%). Lack of appropriate mentorship was listed as second most reason (25.4%). More concerns for not performing cervical endoscopic approaches were the perception of lack of technology (20.8%) and suitable surgical indication (12.5%). Only 4.2% considered cervical endoscopy too risky. Nearly a third (30.6%) of the spine surgeons treated over 80% of their cervical spine patients with endoscopic surgeries. Most commonly performed were posterior endoscopic cervical discectomy (PECD; 52%), posterior endoscopic cervical foraminotomy (PECF; 48%), anterior endoscopic cervical discectomy (AECD; 32%), cervical endoscopic unilateral laminotomy for bilateral decompression (CE-ULBD; 30%), respectively. CONCLUSION: Cervical endoscopic spine surgery is gaining traction among spine surgeons. However, by far most surgeons performing cervical endoscopic spine surgery work in private practice and are autodidacts. This lack of a teacher to shorten the learning curve as well as fear of complications are two of the major impediments to the successful implementation of cervical endoscopic procedures.

Comparison of the Tubular Approach and Uniportal Interlaminar Full-Endoscopic Approach in the Treatment of Lumbar Spinal Stenosis: Our 3-Year Results.

BACKGROUND: To report the long-term results of patients with lumbar spinal stenosis (LSS), for whom we applied the tubular and endoscopic approaches and previously published the short-term results. METHODS: A multicenter, prospective, randomized, double-blind study was carried out to evaluate 2 groups of patients with LSS who underwent microsurgery via a tubular retractor with a unilateral approach (T group) and bilateral spinal decompression using uniportal interlaminar endoscopic approaches (E group). Dural sac cross-sectional and spinal canal cross-sectional areas were measured with the patients' preoperative and postoperative magnetic resonance images. The visual analog scale, Oswestry Disability Index, and Japanese Orthopedic Association scores in the preoperative period and the first, second, and third years after surgery were evaluated. RESULTS: Twenty patients met the inclusion criteria for the research (T group; n = 10, E group; n = 10). The groups' visual analog scale (respectively; P = 0.315, P = 0.529, and P = 0.853), Oswestry Disability Index (respectively; P = 0.529, P = 0.739, and P = 0.912), and Japanese Orthopedic Association (respectively; P = 0.436, P =0.853, and P = 0.684) scores from the first, second, and third postoperative years were quite good compared with the preoperative period, but there was no statistically significant difference. A significant difference was found in the E group, with less blood loss (P < 0.001). CONCLUSIONS: The long-term results of the patients with LSS treated with tubular and endoscopic approaches were similar and very good. Bilateral decompression with minimally invasive spinal surgery methods can be completed with less tissue damage, complications, and blood loss with the unilateral approach.

Full-endoscopic spine surgery diminishes surgical site infections - a propensity score-matched analysis.

BACKGROUND CONTEXT: Surgical site infections (SSI) are one the most frequent and costly complications following spinal surgery. The SSI rates of different surgical approaches need to be analyzed to successfully minimize SSI occurrence. PURPOSE: The purpose of this study was to define the rate of SSIs in patients undergoing full-endoscopic spine surgery (FESS) and then to compare this rate against a propensity score-matched cohort from the National Surgical Quality Improvement Program (NSQIP) database. DESIGN: This is a retrospective multicenter cohort study using a propensity score-matched analysis of prospectively maintained databases. PATIENT SAMPLE: A total of 1277 noninstrumented FESS cases between 2015 and 2021 were selected for analysis. In the nonendoscopic NSQIP cohort we selected data of 55,882 patients. OUTCOME MEASURES: The occurrence of any SSI was the primary outcome. We also collected any other perioperative complications, demographic data, comorbidities, operative details, history of smoking, and chronic steroid intake. METHODS: All FESS cases from a multi-institutional group that underwent surgery from 2015 to 2021 were identified for analysis. A cohort of cases for comparison was identified from the NSQIP database using Current Procedural Terminology of nonendoscopic cervical, thoracic, and lumbar procedures from 2015 to 2019. Trauma cases as well as arthrodesis procedures, surgeries to treat pathologies affecting more than 4 levels or spine tumors that required surgical treatment were excluded. In addition, nonelective cases, and patients with wounds worse than class 1 were also not included. Patient demographics, comorbidities, and operative details were analyzed for propensity matching. RESULTS: In the nonpropensity-matched dataset, the endoscopic cohort had a significantly higher incidence of medical comorbidities. The SSI rates for nonendoscopic and endoscopic patients were 1.2% and 0.001%, respectively, in the nonpropensity match cohort (p-value <.011). Propensity score matching yielded 5936 nonendoscopic patients with excellent matching (standard mean difference of 0.007). The SSI rate in the matched population was 1.1%, compared to 0.001% in endoscopic patients with an odds ratio 0.063 (95% confidence interval (CI) 0.009-0.461, p=.006) favoring FESS. CONCLUSIONS: FESS compares favorably for risk reduction in SSI following spinal decompression surgeries with similar operative characteristics. As a consequence, FESS may be considered the optimal strategy for minimizing SSI morbidity.

DNA extraction from clotted blood in genotyping quality.

DNA extraction from frozen blood clots is challenging. Here, the authors applied QIAGEN Clotspin Baskets and the Gentra Puregene Blood Kit for DNA extraction to cellular fraction of 5.5 ml whole blood without anticoagulating additives. The amount and quality of extracted DNA were assessed via spectrophotometer and gel electrophoresis. Results from array-based genotyping were analyzed. All steps were compared with DNA isolated from anticoagulated blood samples from a separate study. The quality and concentration of DNA extracted from clotted blood were comparable to those of DNA extracted from anticoagulated blood. DNA yield was on average 27 µg per ml clotted blood, with an average purity of 1.87 (A260/A280). Genotyping quality was similar for both DNA sources (call rate: 99.56% from clotted vs 99.49% from anticoagulated blood).

Endoscopic Techniques for Spinal Oncology: A Systematic Literature Review.

BACKGROUND: Due to its ultraminimally invasive nature, endoscopic spinal surgery is an attractive tool in spinal oncologic care. To date, there has been no comprehensive review of this topic. The authors therefore present a thorough search of the medical literature on endoscopic techniques for spinal oncology. METHODS: A systematic review using PubMed was conducted using the following keywords: endoscopic spine surgery, spinal oncology, and spinal tumors. RESULTS: Collectively, 19 cases described endoscopic spine surgery for spinal oncologic care. Endoscopic spine surgery has been employed for the care of patients with spinal tumors under the following 4 circumstances: (1) to obtain a reliable tissue diagnosis; (2) to serve as an adjunct during traditional open surgery; (3) to achieve targeted debulking; or (4) to perform definitive resection. These cases employing endoscopic techniques highlight the versatility of this approach and its utility when applied to the right patient and with an experienced surgeon. CONCLUSIONS: Our systematic review suggests that, given the right patient and an experienced surgeon, endoscopic spine surgery should be considered in the armamentarium for spinal oncologic care for both staging and definitive resection. CLINICAL RELEVANCE: This systematic literature review showed that endoscopic techniques have been successfully applied across the spectrum of care in spinal oncology, from diagnosis to definitive treatment.

Efficacy and Synergy with Cisplatin of an Adenovirus Vectored Therapeutic E1E2E6E7 Vaccine against HPV Genome-Positive C3 Cancers in Mice.

Human papillomavirus (HPV) infections are the main cause of cervical and oropharyngeal cancers. As prophylactic vaccines have no curative effect, an efficient therapy would be highly desired. Most therapeutic vaccine candidates target only a small subset of HPV regulatory proteins, namely, E6 and E7, and are therefore restricted in the breadth of their immune response. However, research has suggested E1 and E2 as promising targets to fight HPV+ cancer. Here, we report the design of adenoviral vectors efficiently expressing HPV16 E1 and E2 in addition to transformation-deficient E6 and E7. Vaccination elicited vigorous CD4+ and CD8+ T-cell responses against all encoded HPV16 proteins in outbred mice and against E1 and E7 in C57BL/6 mice. Therapeutic vaccination of C3 tumor-bearing mice led to significantly reduced tumor growth and enhanced survival for both small and established tumors. Tumor biopsies revealed increased numbers of tumor-infiltrating CD8+ T cells in treated mice. Cisplatin enhanced the effect of therapeutic vaccination, accompanied by enhanced infiltration of dendritic cells into the tumor. CD8+ T cells were identified as effector cells in T-cell depletion assays, seemingly under regulation by FoxP3+CD4+ regulatory T cells. Finally, therapeutic vaccination with Ad-Ii-E1E2E6E7 exhibited significantly enhanced survival compared with vaccination with two peptides each harboring a known E6/E7 epitope. We hypothesize that this difference could be due to the induction of additional T-cell responses against E1. These results support the use of this novel vaccine candidate targeting an extended set of antigens (Ad-Ii-E1E2E6E7), in combination with cisplatin, as an advanced strategy to combat HPV+ cancers.

Higher Infection Risk among Health Care Workers and Lower Risk among Smokers Persistent across SARS-CoV-2 Waves-Longitudinal Results from the Population-Based TiKoCo Seroprevalence Study.

SARS-CoV-2 seroprevalence was reported as substantially increased in medical personnel and decreased in smokers after the first wave in spring 2020, including in our population-based Tirschenreuth Study (TiKoCo). However, it is unclear whether these associations were limited to the early pandemic and whether the decrease in smokers was due to reduced infection or antibody response. We evaluated the association of occupation and smoking with period-specific seropositivity: for the first wave until July 2020 (baseline, BL), the low infection period in summer (follow-up 1, FU1, November 2020), and the second/third wave (FU2, April 2021). We measured binding antibodies directed to SARS-CoV-2 nucleoprotein (N), viral spike protein (S), and neutralizing antibodies at BL, FU1, and FU2. Previous infection, vaccination, smoking, and occupation were assessed by questionnaires. The 4181 participants (3513/3374 at FU1/FU2) included 6.5% medical personnel and 20.4% current smokers. At all three timepoints, new seropositivity was higher in medical personnel with ORs = 1.99 (95%-CI = 1.36-2.93), 1.41 (0.29-6.80), and 3.17 (1.92-5.24) at BL, FU1, and FU2, respectively, and nearly halved among current smokers with ORs = 0.47 (95%-CI = 0.33-0.66), 0.40 (0.09-1.81), and 0.56 (0.33-0.94). Current smokers compared to never-smokers had similar antibody levels after infection or vaccination and reduced odds of a positive SARS-CoV-2 result among tested. Our data suggest that decreased seroprevalence among smokers results from fewer infections rather than reduced antibody response. The persistently higher infection risk of medical staff across infection waves, despite improved means of protection over time, underscores the burden for health care personnel.

Early and Long-Term HIV-1 Immunogenicity Induced in Macaques by the Combined Administration of DNA, NYVAC and Env Protein-Based Vaccine Candidates: The AUP512 Study.

To control HIV infection there is a need for vaccines to induce broad, potent and long-term B and T cell immune responses. With the objective to accelerate and maintain the induction of substantial levels of HIV-1 Env-specific antibodies and, at the same time, to enhance balanced CD4 and CD8 T cell responses, we evaluated the effect of concurrent administration of MF59-adjuvanted Env protein together with DNA or NYVAC vectors at priming to establish if early administration of Env leads to early induction of antibody responses. The primary goal was to assess the immunogenicity endpoint at week 26. Secondary endpoints were (i) to determine the quality of responses with regard to RV144 correlates of protection and (ii) to explore a potential impact of two late boosts. In this study, five different prime/boost vaccination regimens were tested in rhesus macaques. Animals received priming immunizations with either NYVAC or DNA alone or in combination with Env protein, followed by NYVAC + protein or DNA + protein boosts. All regimens induced broad, polyfunctional and well-balanced CD4 and CD8 T cell responses, with DNA-primed regimens eliciting higher response rates and magnitudes than NYVAC-primed regimens. Very high plasma binding IgG titers including V1/V2 specific antibodies, modest antibody-dependent cellular cytotoxicity (ADCC) and moderate neutralization activity were observed. Of note, early administration of the MF59-adjuvanted Env protein in parallel with DNA priming leads to more rapid elicitation of humoral responses, without negatively affecting the cellular responses, while responses were rapidly boosted after repeated immunizations, indicating the induction of a robust memory response. In conclusion, our findings support the use of the Env protein component during priming in the context of an heterologous immunization regimen with a DNA and/or NYVAC vector as an optimized immunization protocol against HIV infection.

Directing HIV-1 for degradation by non-target cells, using bi-specific single-chain llama antibodies.

While vaccination against HIV-1 has been so far unsuccessful, recently broadly neutralizing antibodies (bNAbs) against HIV-1 envelope glycoprotein were shown to induce long-term suppression in the absence of antiretroviral therapy in patients with antibody-sensitive viral reservoirs. The requirement of neutralizing antibodies indicates that the antibody mediated removal (clearance) of HIV-1 in itself is not efficient enough in these immune compromised patients. Here we present a novel, alternative approach that is independent of a functional immune system to clear HIV-1, by capturing the virus and redirecting it to non-target cells where it is internalized and degraded. We use bispecific antibodies with domains derived from small single chain Llama antibodies (VHHs). These bind with one domain to HIV-1 envelope proteins and with the other domain direct the virus to cells expressing epidermal growth factor receptor (EGFR), a receptor that is ubiquitously expressed in the body. We show that HIV envelope proteins, virus-like particles and HIV-1 viruses (representing HIV-1 subtypes A, B and C) are efficiently recruited to EGFR, internalized and degraded in the lysosomal pathway at low nM concentrations of bispecific VHHs. This directed degradation in non-target cells may provide a clearance platform for the removal of viruses and other unwanted agents from the circulation, including toxins, and may thus provide a novel method for curing.

Full Endoscopic Surgery for Thoracic Pathology: Next Step after Mastering Lumbar and Cervical Endoscopic Spine Surgery?

Thoracic disc herniation and stenosis are relatively rare, and various symptoms make them difficult to diagnose. Due to the complexity of neural and vascular structure, surgical treatment of thoracic pathology is challenging. Endoscopic spine surgery is an emerging minimally invasive surgical option. Based on wide experience on the cervical and lumbar spine, an endoscopic approach for the thoracic pathology can be performed beyond the learning curve. Transforaminal approach for thoracic disc herniation, endoscopic unilateral approach, and bilateral decompression for thoracic stenosis have been reported as favorable and safe surgical options. In the present study, the authors described the detailed surgical procedure as well as tips and tricks.

Design and Immunological Validation of Macaca fascicularis Papillomavirus Type 3 Based Vaccine Candidates in Outbred Mice: Basis for Future Testing of a Therapeutic Papillomavirus Vaccine in NHPs.

Persistent human papillomavirus (HPV) infections are causative for cervical neoplasia and carcinomas. Despite the availability of prophylactic vaccines, morbidity and mortality induced by HPV are still too high. Thus, an efficient therapy, such as a therapeutic vaccine, is urgently required. Herein, we describe the development and validation of Macaca fascicularis papillomavirus type 3 (MfPV3) antigens delivered via nucleic-acid and adenoviral vectors in outbred mouse models. Ten artificially fused polypeptides comprising early viral regulatory proteins were designed and optionally linked to the T cell adjuvant MHC-II-associated invariant chain. Transfected HEK293 cells and A549 cells transduced with recombinant adenoviruses expressing the same panel of artificial antigens proved proper and comparable expression, respectively. Immunization of outbred CD1 and OF1 mice led to CD8+ and CD4+ T cell responses against MfPV3 antigens after DNA- and adenoviral vector delivery. Moreover, in vivo cytotoxicity of vaccine-induced CD8+ T cells was demonstrated in BALB/c mice by quantifying specific killing of transferred peptide-pulsed syngeneic target cells. The use of the invariant chain as T cell adjuvant enhanced the T cell responses regarding cytotoxicity and in vitro analysis suggested an accelerated turnover of the antigens as causative. Notably, the fusion-polypeptide elicited the same level of T-cell responses as administration of the antigens individually, suggesting no loss of immunogenicity by fusing multiple proteins in one vaccine construct. These data support further development of the vaccine candidates in a follow up efficacy study in persistently infected Macaca fascicularis monkeys to assess their potential to eliminate pre-malignant papillomavirus infections, eventually instructing the design of an analogous therapeutic HPV vaccine.

Estimates and Determinants of SARS-Cov-2 Seroprevalence and Infection Fatality Ratio Using Latent Class Analysis: The Population-Based Tirschenreuth Study in the Hardest-Hit German County in Spring 2020.

SARS-CoV-2 infection fatality ratios (IFR) remain controversially discussed with implications for political measures. The German county of Tirschenreuth suffered a severe SARS-CoV-2 outbreak in spring 2020, with particularly high case fatality ratio (CFR). To estimate seroprevalence, underreported infections, and IFR for the Tirschenreuth population aged ≥14 years in June/July 2020, we conducted a population-based study including home visits for the elderly, and analyzed 4203 participants for SARS-CoV-2 antibodies via three antibody tests. Latent class analysis yielded 8.6% standardized county-wide seroprevalence, a factor of underreported infections of 5.0, and 2.5% overall IFR. Seroprevalence was two-fold higher among medical workers and one third among current smokers with similar proportions of registered infections. While seroprevalence did not show an age-trend, the factor of underreported infections was 12.2 in the young versus 1.7 for ≥85-year-old. Age-specific IFRs were <0.5% below 60 years of age, 1.0% for age 60-69, and 13.2% for age 70+. Senior care homes accounted for 45% of COVID-19-related deaths, reflected by an IFR of 7.5% among individuals aged 70+ and an overall IFR of 1.4% when excluding senior care home residents from our computation. Our data underscore senior care home infections as key determinant of IFR additionally to age, insufficient targeted testing in the young, and the need for further investigations on behavioral or molecular causes of the fewer infections among current smokers.

Augmenting the Immune Response against a Stabilized HIV-1 Clade C Envelope Trimer by Silica Nanoparticle Delivery.

The delivery of HIV-1 envelope (Env) trimer-based immunogens on the surface of nanoparticles holds promise to promote immunogenicity with the aim of inducing a potent, durable and broad neutralizing antibody (bnAb) response. Towards that goal, we examined the covalent conjugation of Env to 100 nm and 200 nm silica nanoparticles (SiNPs) to optimize conjugation density and attachment stability. Env was redesigned to enable site-specific cysteine-mediated covalent conjugation while maintaining its structural integrity and antigenicity. Env was anchored to different sized SiNPs with a calculated spacing of 15 nm between adjacent trimers. Both particle sizes exhibited high in vitro stability over a seven-day period. After attachment, 100 nm particles showed better colloidal stability compared to 200 nm particles. Importantly, the antigenic profile of Env was not impaired by surface attachment, indicating that the quaternary structure was maintained. In vitro Env uptake by dendritic cells was significantly enhanced when Env was delivered on the surface of nanoparticles compared to soluble Env. Furthermore, multivalent Env displayed efficiently activated B cells even at Env concentrations in the low nanomolar range. In mice, antibody responses to nanoparticle-coupled Env were stronger compared to the free protein and had equivalent effects at lower doses and without adjuvant.

A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response.

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and ß-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small ß-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The ß-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the ß-annulus peptide for vaccine design.