RelCoVax®, a two antigen subunit protein vaccine candidate against SARS-CoV-2 induces strong immune responses in mice

The COVID-19 pandemic has spurred an unprecedented movement to develop safe and effective vaccines against the SARS-CoV-2 virus to immunize the global population. The first set of vaccine candidates that received emergency use authorization targeted the spike (S) glycoprotein of the SARS-CoV-2 virus that enables virus entry into cells via the receptor binding domain (RBD). Recently, multiple variants of SARS-CoV-2 have emerged with mutations in S protein and the ability to evade neutralizing antibodies in vaccinated individuals. We have developed a dual RBD and nucleocapsid (N) subunit protein vaccine candidate named RelCoVax® through heterologous expression in mammalian cells (RBD) and E. coli (N). The RelCoVax® formulation containing a combination of aluminum hydroxide (alum) and a synthetic CpG oligonucleotide as adjuvants elicited high antibody titers against RBD and N proteins in mice after a prime and boost dose regimen administered 2 weeks apart. The vaccine also stimulated cellular immune responses with a potential Th1 bias as evidenced by increased IFN-γ release by splenocytes from immunized mice upon antigen exposure particularly N protein. Finally, the serum of mice immunized with RelCoVax® demonstrated the ability to neutralize two different SARS-CoV-2 viral strains in vitro including the Delta strain that has become dominant in many regions of the world and can evade vaccine induced neutralizing antibodies. These results warrant further evaluation of RelCoVax® through advanced studies and contribute towards enhancing our understanding of multicomponent subunit vaccine candidates against SARS-CoV-2.

Safety, Tolerability and Immunogenicity of Oxford-AstraZeneca ChAdOx1 Vaccine Among Polish School-Teachers

Background Polish teachers, as the priority group, were offered the ChAdOx1-S vaccine since February 2021. The objective was to investigate safety, tolerability and immunogenicity of this vaccine following two vaccine doses. Methods Teachers were invited for serological testing ≥8 weeks after second vaccination. Quantitative post-vaccination anti-spike antibody responses were measured using the Abbott SARS-CoV-2 IgG II Quant assay (detection threshold: ≥7.1 BAU/ml). Multivariable logistic regression methods were used to identify predictors of immunogenicity. Results Of 192 teachers, mean age 50.5±8.3 years, 83.9% were females. Median (range) dosing interval was 50 (14-95) days; median interval between the second dose and immunogenicity test was 69 days (range: 57–111). More than a half of teachers (58.3%) reported they would change the product for another (mostly mRNA) vaccine if there was such an opportunity. Adverse reactions after receiving the vaccine (either the first or the second dose) were reported by 79.2% teachers, more frequently after the first dose (84.9%), and were similar in nature to those previously reported: feeling feverish (44.8%), headache (41.7%), malaise, chills (both: 38.0%), injection-site tenderness (37.5%) and pain (32.3%). Less males than females (54.8% vs 80.1%) and older (aged ≥50 years) than younger teachers (65.7% vs 90.4%) reported side effects (p<0.002; p<0.0001, respectively). By ≥8 weeks after the boost dose, all teachers had neutralizing antibody responses. The median (range) anti-spike IgG reading was 525.0 BAU/mL (20.6-5680.0 BAU/mL); 1008.02 (115.3–5680.0) BAU/mL in teachers with evidence of prior infection and 381.42 BAU/mL (20.6–3108.8) in those without (p=0.001). Previous infection with SARS-CoV-2 and longer dose interval were both positive predictors of higher immunologic response (p<0.0001; p=0.01, respectively), with no evidence of differences by age, gender, BMI, smoking or comorbidities. Conclusions The results demonstrated good safety, tolerability and immunogenicity of the ChAdOx1-S vaccine. Immunization led to detectable anti-spike antibodies in all teachers. Our study justifies the longer dose interval as an important factor to enhance higher antibody response. Findings suggest that in immunocompetent vaccine recipients with an evidence of previous infection a delay regarding the second dose could be considered when careful management in the use of vaccine resources is needed.

Real-world serologic responses to Extended-interval and Heterologous COVID-19 mRNA vaccination in Frail Elderly - Interim report from a prospective observational cohort study

BackgroundThe Coronavirus disease 2019 (Covid-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has prompted accelerated vaccines development. Their use was prioritized to protect the most vulnerable, notably, the elderly. Because of fluctuations in vaccine availability, strategies such as delayed second dose and heterologous prime-boost have been employed. The effectiveness of these strategies in the frail elderly are unknown.MethodsIn this real-world vaccination study, under a government-decreed rationing strategy, elderly adults residing in long-term care facilities, with or without previously-documented SARS-CoV-2 infection, were administered homologous or heterologous mRNA vaccines, with an extended 16-week interval between doses. Clinical data and blood were serially collected during and after this interval period. Sera were tested for SARS-CoV-2-specific IgG antibodies (to trimeric S; RBD; nucleocapsid) by automated chemiluminescent ELISA.FindingsAfter a significant increase 4 weeks post-prime dose, there was a significant decline in anti-RBD and anti-S IgG levels until the boost dose, followed by an increase 4 weeks later. Previously uninfected individuals exhibited lower antibody responses up to 16 weeks post-prime dose, but achieved comparable levels to previously infected counterparts by 4 weeks post-second dose. Individuals primed with BNT162b2 exhibited larger decrease in anti-RBD and anti-S IgG levels with 16-week interval between doses, compared to those who received mRNA-1273. No differences in antibody levels 4 weeks after the second dose were noted between the two vaccines, in either homologous or heterologous combinations.InterpretationsThese interim results of this ongoing longitudinal study show that, among frail elderly, neither age, sex, nor comorbidity affect antigenicity of mRNA-based COVID vaccines, but previous SARS-CoV-2 infection and type of mRNA vaccine influenced antibody responses when used with a 16-week interval between doses. Homologous/heterologous use of mRNA vaccines was not associated with significant differences in antibody responses 4 weeks following second dose, supporting their interchangeability.FundingThis project was supported by funding from the Public Health Agency of Canada, through the Vaccine Surveillance Reference group and the COVID-19 Immunity Task Force (CITF).

Novel lipid nanoparticle provides potent SARS-CoV-2 mRNA vaccine at low dose with low local reactogenicity, high thermostability and limited systemic biodistribution

Concerns with current mRNA Lipid Nanoparticle (LNP) systems include dose-limiting reactogenicity, adverse events that may be partly due to systemic off target expression of the immunogen, and a very limited understanding of the mechanisms responsible for the frozen storage requirement. We applied a new rational design process to identify a novel multiprotic ionizable lipid, called C24, as the key component of the mRNA LNP delivery system. We show that the resulting C24 LNP has a multistage protonation behavior resulting in greater endosomal protonation and greater translation of an mRNA-encoded luciferase reporter after intramuscular (IM) administration compared to the standard reference MC3 LNP. Off-target expression in liver after IM administration was reduced 6 fold for the C24 LNP compared to MC3. Neutralizing titers in immunogenicity studies delivering a nucleoside-modified mRNA encoding for the diproline stabilized spike protein immunogen were 10 fold higher for the C24 LNP versus MC3, and protection against viral challenge in a SARS-CoV-2 mouse model occurred at a very low 0.25 µg prime/boost dose of the same immunogen in the C24 LNP. Injection site inflammation was notably reduced for C24 compared to MC3. In addition, we found the C24 LNP to be entirely stable in bioactivity and mRNA integrity when stored at 4 ºC for at least 19 days. Storage at higher temperatures reduced both bioactivity and mRNA integrity, but less so for C24 than MC3, and in a manner consistent with the phosphodiester transesterification reaction mechanism of mRNA cleavage. The higher potency, lower injection site inflammation, and higher stability of the C24 LNP present important advancements in the evolution mRNA vaccine delivery.

Assessment of low-intensity transcranial magnetic stimulation (TMS) in treatment for high-grade glioma (HGG).

e14027 Background: The existing modern standards of combination treatment of HGG patients do not provide recovery and a long-term favorable prognosis, and the increasong incidence of HGG determines the need for additional effective technologies for anticancer and decongestant therapy. One of such methods involves TMS, and we have reported its preliminary assessment earlier (DOI: 10.1200/JCO.2020.38.15_suppl.2545). In this study, we continued the observation to examine the survival of patients. Methods: Patients with HGG received combination treatment: stage 1 – surgical removal of tumors within visible unaltered tissues; stage 2 – radiation therapy (the Varian Novalis linear accelerator) to the bed of the removed tumor, single boost dose 2 Gy, total boost dose 60 Gy; stage 3 – multi-course chemotherapy: temozolomide 150 mg/m² on days 1-5 with a 23-day interval. Starting from the second day after surgery, patients of group 1 (n = 25) received 10 TMS sessions, and during radiotherapy – 15 TMS sessions. Patients of group 2 (n = 25) received combination treatment without TMS. 6 and 12 months after the surgery, survival of patients was assessed with the Kaplan-Meier method and the Log-Rank test. Results: After 6 months of the follow-up, the survival of patients in group 1 remained at 100%, while in the control group it decreased to 88.8±8.7%. The difference in the 1-year overall survival was even more pronounced: in group 1, it was 68.5±10.4%, exceeding the value in group 2 (52.0±7.5%.) The differences were statistically significant (Log-Rank test p = 0.001). Conclusions: The results confirm the effectiveness of accompanying TMS in the early postoperative period, as well as at the stage of radiation therapy. The undoubted effectiveness of the considered techniques makes it expedient to include this type of treatment in the combination therapy for HGG patients. The reported study was funded by RFBR, project number № 19-315-90082\19.

Comparison of IORT (Radical and Boost Dose) and EBRT in Terms of Disease-Free Survival and Overall Survival according to Demographic, Pathologic, and Biological Factors in Patients with Breast Cancer

Background. The standard treatment for breast cancer is breast-conserving surgery (BCS) with radiotherapy. If external beam radiation therapy (EBRT) can be safely replaced with intraoperative radiotherapy (IORT), it will help patients to save their breast and to have equivocal or better results in DFS and overall survival (OS). Methods. A total of 2022 patients with breast cancer treated during 6 years were enrolled in the current study. A total of 657, 376, and 989 patients received EBRT, radical, and boost dose by IORT, respectively, according to the IRIORT consensus protocol. The primary endpoint was recurrence and death. The secondary endpoint was the role of variables in recurrence and death. Results. With a mean follow-up of 34.5 and 40.18 months for the IORT and EBRT groups, respectively, there was a significant difference in DFS between electron boost and X-ray boost groups ( P = 0.037 ) and the electron radical group compared with EBRT ( P = 0.025 ), but there was no significant difference between other boost and radical groups in DFS and OS. Conclusions. IORT can be a preferred treatment modality because of its noninferior outcomes, and in some special conditions, it has superior outcomes compared to EBRT, particularly in delivering radical dose with IORT.

Patient-Derived Nasopharyngeal Cancer Organoids for Disease Modeling and Radiation Dose Optimization

Effective radiation treatment (RT) for recurrent nasopharyngeal cancers (NPC), featuring an intrinsic hypoxic sub-volume, remains a clinical challenge. Lack of disease‐specific in-vitro models of NPC, together with difficulties in establishing patient derived xenograft (PDX) models, have further hindered development of personalized therapeutic options. Herein, we established two NPC organoid lines from recurrent NPC PDX models and further characterized and compared these models with original patient tumors using RNA sequencing analysis. Organoids were cultured in hypoxic conditions to examine the effects of hypoxia and radioresistance. These models were then utilized to determine the radiobiological parameters, such as α/β ratio and oxygen enhancement ratio (OER), characteristic to radiosensitive normoxic and radioresistant hypoxic NPC, using simple dose-survival data analytic tools. The results were further validated in-vitro and in-vivo, to determine the optimal boost dose and fractionation regimen required to achieve effective NPC tumor regression. Despite the differences in tumor microenvironment due to the lack of human stroma, RNA sequencing analysis revealed good correlation of NPC PDX and organoid models with patient tumors. Additionally, the established models also mimicked inter-tumoral heterogeneity. Hypoxic NPC organoids were highly radioresistant and had high α/β ratio compared to its normoxic counterparts. In-vitro and in-vivo fractionation studies showed that hypoxic NPC was less sensitive to RT fractionation scheme and required a large bolus dose or 1.4 times of the fractionated dose that was effective against normoxic cells in order to compensate for oxygen deficiency. This study is the first direct experimental evidence to predict optimal RT boost dose required to cause sufficient damage to recurrent hypoxic NPC tumor cells, which can be further used to develop dose-painting algorithms in clinical practice.

Effect of a single or two doses of an anti-GnRH vaccine on testicle morpho-functional characteristics in Nelore bulls

The aim of this study was to compare testicle morpho-functional characteristics in bulls undergoing a single or two immunizations against GnRH. Nelore (Bos taurus indicus) bulls were randomly allocated into three experimental groups: G1 (n=12), a single 400 μg dose of anti-GnRH vaccine on day 0; G2 (n=11), a first 400 μg dose of anti-GnRH vaccine on day 0 followed by a second (boost) dose 30 days later; and control group (CG, n=12), 1 mL saline 0.9% at day 0. Every 30 days, from day 0 until slaughter at day 90, the bulls were weighed and underwent testicular biometry, semen collection and analysis, and blood sample collection for testosterone measurement. Immediately after slaughter, the testicles were removed and transport at 15°C to the laboratory for histopathological analysis. There was a decrease in testicular height (P=0.0476), width (P=0.0021), and in scrotal circumference (P=0.0001), after either a single (G1) or two (G2) immunizations against GnRH. Both G1 and G2 had lower testosterone concentrations than CG from day 60 on (P<0.01), but in G2, it was also lower than in G1 at day 90 (P=0.0006). All sperm parameters were affected by active immunization against GnRH (P<0.05), and in G2, averages were lesser (P<0.05) than in G1 from day 60 on. No signs of seminiferous tubule degeneration were found in any sample from the CG, contrasting with 75.0% and 100.0% of the samples from G1 and G2, respectively. In summary, immunocastration affected testicle morpho-functional characteristics in bulls in a time- and dose-dependent way.

mRNA-1273 efficacy in a severe COVID-19 model: attenuated activation of pulmonary immune cells after challenge

ABSTRACTThe mRNA-1273 vaccine was recently determined to be effective against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from interim Phase 3 results. Human studies, however, cannot provide the controlled response to infection and complex immunological insight that are only possible with preclinical studies. Hamsters are the only model that reliably exhibit more severe SARS-CoV-2 disease similar to hospitalized patients, making them pertinent for vaccine evaluation. We demonstrate that prime or prime-boost administration of mRNA-1273 in hamsters elicited robust neutralizing antibodies, ameliorated weight loss, suppressed SARS-CoV-2 replication in the airways, and better protected against disease at the highest prime-boost dose. Unlike in mice and non-human primates, mRNA-1273- mediated immunity was non-sterilizing and coincided with an anamnestic response. Single-cell RNA sequencing of lung tissue permitted high resolution analysis which is not possible in vaccinated humans. mRNA-1273 prevented inflammatory cell infiltration and the reduction of lymphocyte proportions, but enabled antiviral responses conducive to lung homeostasis. Surprisingly, infection triggered transcriptome programs in some types of immune cells from vaccinated hamsters that were shared, albeit attenuated, with mock-vaccinated hamsters. Our results support the use of mRNA-1273 in a two-dose schedule and provides insight into the potential responses within the lungs of vaccinated humans who are exposed to SARS-CoV-2.

Evidence For COVID-19 Vaccine Deferred Dose 2 Boost Timing

Both Pfizer and Moderna vaccine trials used accelerated methods to achieve rapid FDA approval with minimal and wise compromises. While many established vaccines use boost / dose 2 timings that are typically in the 3 to 120 months range , dose 2 timings for both trials were less than 30 days as part of a crucial effort to rapidly develop a vaccine with significant efficacy. Data from both trials showed excellent dose 1 efficacy; however, neither of the related Safety and Efficacy publications highlighted this dose 1 efficacy excellence.The trials, manufacturing, and distribution programs have rapidly delivered millions of doses to points of care. However, the ability to administer these doses at localities as rapidly as they can be distributed has been shown as the largest current challenge to achieving the widespread vaccination. Improvements could reduce mortality rates that presently exceed 2000 per day, avoid serious cases at a time where hospital census is at full capacity or beyond, and minimize long-term sequelae.This study suggests that it is possible to achieve these and other goals by recognizing the evidence for COVID-19 vaccine deferred boost timing and using a more conventional, decades-tested, boost timing on the order of months. This would enable currently limited US resources to effectively double the population being vaccinated over the next few months, enabling more rapid vaccination of those at highest-risk for severe infections.