Immunologic predictors of vaccine responsiveness in patients with lymphoma and CLL.

Patients with B-cell lymphomas have altered cellular components of vaccine responses due to malignancy and therapy, and the optimal timing of vaccination relative to therapy remains unknown. SARS-CoV-2 vaccines created an opportunity for new insights in vaccine timing because patients were challenged with a novel antigen across multiple phases of treatment. We studied serologic mRNA vaccine response in retrospective and prospective cohorts with lymphoma and CLL, paired with clinical and research immune parameters. Reduced serologic response was observed more frequently during active therapies, but non-response was also common within observation and post-treatment groups. Total IgA and IgM correlated with successful vaccine response. In individuals treated with CART-19, non-response was associated with reduced B and T follicular helper cells. Predictors of vaccine response varied by disease and therapeutic group, and therefore further studies of immune health during and after cancer therapies are needed to allow individualized vaccine timing.

Safety of an Alternative Care Protocol for EMS Non-Transport in the COVID-19 Pandemic.

AIM: Our primary goal was to evaluate safety of a new emergency medical services (EMS) protocol directing non-transport of low-acuity patients during the COVID-19 pandemic. METHODS: We performed a retrospective cohort analysis of all patients in Marion County, Indiana, from March 23, 2020 to May 25, 2020 for whom a novel non-transport protocol was used by EMS for patients with low-acuity COVID-19 symptoms. We assessed paramedic compliance with the protocol to determine numbers and types of deviations. We further reviewed a statewide health information exchange database to identify any patients with emergency department (ED) visits, hospital admissions, or death within 30 days of the EMS non-transport. For ED and hospital visits, we collected ED or admission diagnoses to determine if the etiologies were COVID-related. RESULTS: Between March 24, 2020 and May 25, 2020, 222 patients were documented as "Treated, Released (per protocol)." The protocol was correctly applied 144 times (64.8%). The other 78 times, although the EMS clinicians documented use of the protocol, it was not actually used (e.g., another protocol such as "no medical emergency" was used). Of the 144 patients for whom the protocol was used, in 55 cases (38.2%), the clinicians documented patient factors that should have contraindicated use of the protocol (e.g., chest pain, past medical history of asthma). The protocol was applied 5 times (3.5%) in pediatric patients. Two patients were admitted to the hospital within 72 hours of incorrect application of the protocol; both were for COVID-related complaints. Two patients were admitted to the hospital within 72 hours of correct protocol use; one was for a COVID-related complaint. CONCLUSION: In this case series, paramedics demonstrated large deviations from the novel non-transport protocol. Several patients were admitted to the hospital within 72 hours of non-transport both when the protocol was used correctly, and when it was used incorrectly.

ActinoBase: tools and protocols for researchers working on Streptomyces and other filamentous actinobacteria.

Actinobacteria is an ancient phylum of Gram-positive bacteria with a characteristic high GC content to their DNA. The ActinoBase Wiki is focused on the filamentous actinobacteria, such as Streptomyces species, and the techniques and growth conditions used to study them. These organisms are studied because of their complex developmental life cycles and diverse specialised metabolism which produces many of the antibiotics currently used in the clinic. ActinoBase is a community effort that provides valuable and freely accessible resources, including protocols and practical information about filamentous actinobacteria. It is aimed at enabling knowledge exchange between members of the international research community working with these fascinating bacteria. ActinoBase is an anchor platform that underpins worldwide efforts to understand the ecology, biology and metabolic potential of these organisms. There are two key differences that set ActinoBase apart from other Wiki-based platforms: [1] ActinoBase is specifically aimed at researchers working on filamentous actinobacteria and is tailored to help users overcome challenges working with these bacteria and [2] it provides a freely accessible resource with global networking opportunities for researchers with a broad range of experience in this field.

Efficient recall of Omicron-reactive B cell memory after a third dose of SARS-CoV-2 mRNA vaccine.

We examined antibody and memory B cell responses longitudinally for ∼9-10 months after primary 2-dose SARS-CoV-2 mRNA vaccination and 3 months after a 3rd dose. Antibody decay stabilized between 6 and 9 months, and antibody quality continued to improve for at least 9 months after 2-dose vaccination. Spike- and RBD-specific memory B cells remained durable over time, and 40%-50% of RBD-specific memory B cells simultaneously bound the Alpha, Beta, Delta, and Omicron variants. Omicron-binding memory B cells were efficiently reactivated by a 3rd dose of wild-type vaccine and correlated with the corresponding increase in neutralizing antibody titers. In contrast, pre-3rd dose antibody titers inversely correlated with the fold-change of antibody boosting, suggesting that high levels of circulating antibodies may limit the added protection afforded by repeat short interval boosting. These data provide insight into the quantity and quality of mRNA-vaccine-induced immunity over time through 3 or more antigen exposures.

Efficient recall of Omicron-reactive B cell memory after a third dose of SARS-CoV-2 mRNA vaccine.

Despite a clear role in protective immunity, the durability and quality of antibody and memory B cell responses induced by mRNA vaccination, particularly by a 3 rd dose of vaccine, remains unclear. Here, we examined antibody and memory B cell responses in a cohort of individuals sampled longitudinally for ∼9-10 months after the primary 2-dose mRNA vaccine series, as well as for ∼3 months after a 3 rd mRNA vaccine dose. Notably, antibody decay slowed significantly between 6- and 9-months post-primary vaccination, essentially stabilizing at the time of the 3 rd dose. Antibody quality also continued to improve for at least 9 months after primary 2-dose vaccination. Spike- and RBD-specific memory B cells were stable through 9 months post-vaccination with no evidence of decline over time, and ∼40-50% of RBD-specific memory B cells were capable of simultaneously recognizing the Alpha, Beta, Delta, and Omicron variants. Omicron-binding memory B cells induced by the first 2 doses of mRNA vaccine were boosted significantly by a 3rd dose and the magnitude of this boosting was similar to memory B cells specific for other variants. Pre-3 rd dose memory B cell frequencies correlated with the increase in neutralizing antibody titers after the 3 rd dose. In contrast, pre-3 rd dose antibody titers inversely correlated with the fold-change of antibody boosting, suggesting that high levels of circulating antibodies may limit reactivation of immunological memory and constrain further antibody boosting by mRNA vaccines. These data provide a deeper understanding of how the quantity and quality of antibody and memory B cell responses change over time and number of antigen exposures. These data also provide insight into potential immune dynamics following recall responses to additional vaccine doses or post-vaccination infections.

mRNA vaccines induce durable immune memory to SARS-CoV-2 and variants of concern.

The durability of immune memory after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) messenger RNA (mRNA) vaccination remains unclear. In this study, we longitudinally profiled vaccine responses in SARS-CoV-2­naïve and ­recovered individuals for 6 months after vaccination. Antibodies declined from peak levels but remained detectable in most subjects at 6 months. By contrast, mRNA vaccines generated functional memory B cells that increased from 3 to 6 months postvaccination, with the majority of these cells cross-binding the Alpha, Beta, and Delta variants. mRNA vaccination further induced antigen-specific CD4+ and CD8+ T cells, and early CD4+ T cell responses correlated with long-term humoral immunity. Recall responses to vaccination in individuals with preexisting immunity primarily increased antibody levels without substantially altering antibody decay rates. Together, these findings demonstrate robust cellular immune memory to SARS-CoV-2 and its variants for at least 6 months after mRNA vaccination.

Rapid induction of antigen-specific CD4+ T cells is associated with coordinated humoral and cellular immunity to SARS-CoV-2 mRNA vaccination.

SARS-CoV-2 mRNA vaccines have shown remarkable clinical efficacy, but questions remain about the nature and kinetics of T cell priming. We performed longitudinal antigen-specific T cell analyses on healthy SARS-CoV-2-naive and recovered individuals prior to and following mRNA prime and boost vaccination. Vaccination induced rapid antigen-specific CD4+ T cell responses in naive subjects after the first dose, whereas CD8+ T cell responses developed gradually and were variable in magnitude. Vaccine-induced Th1 and Tfh cell responses following the first dose correlated with post-boost CD8+ T cells and neutralizing antibodies, respectively. Integrated analysis revealed coordinated immune responses with distinct trajectories in SARS-CoV-2-naive and recovered individuals. Last, whereas booster vaccination improved T cell responses in SARS-CoV-2-naive subjects, the second dose had little effect in SARS-CoV-2-recovered individuals. These findings highlight the role of rapidly primed CD4+ T cells in coordinating responses to the second vaccine dose in SARS-CoV-2-naive individuals.

mRNA Vaccination Induces Durable Immune Memory to SARS-CoV-2 with Continued Evolution to Variants of Concern.

SARS-CoV-2 mRNA vaccines have shown remarkable efficacy, especially in preventing severe illness and hospitalization. However, the emergence of several variants of concern and reports of declining antibody levels have raised uncertainty about the durability of immune memory following vaccination. In this study, we longitudinally profiled both antibody and cellular immune responses in SARS-CoV-2 naïve and recovered individuals from pre-vaccine baseline to 6 months post-mRNA vaccination. Antibody and neutralizing titers decayed from peak levels but remained detectable in all subjects at 6 months post-vaccination. Functional memory B cell responses, including those specific for the receptor binding domain (RBD) of the Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) variants, were also efficiently generated by mRNA vaccination and continued to increase in frequency between 3 and 6 months post-vaccination. Notably, most memory B cells induced by mRNA vaccines were capable of cross-binding variants of concern, and B cell receptor sequencing revealed significantly more hypermutation in these RBD variant-binding clones compared to clones that exclusively bound wild-type RBD. Moreover, the percent of variant cross-binding memory B cells was higher in vaccinees than individuals who recovered from mild COVID-19. mRNA vaccination also generated antigen-specific CD8+ T cells and durable memory CD4+ T cells in most individuals, with early CD4+ T cell responses correlating with humoral immunity at later timepoints. These findings demonstrate robust, multi-component humoral and cellular immune memory to SARS-CoV-2 and current variants of concern for at least 6 months after mRNA vaccination. Finally, we observed that boosting of pre-existing immunity with mRNA vaccination in SARS-CoV-2 recovered individuals primarily increased antibody responses in the short-term without significantly altering antibody decay rates or long-term B and T cell memory. Together, this study provides insights into the generation and evolution of vaccine-induced immunity to SARS-CoV-2, including variants of concern, and has implications for future booster strategies.

Distinct antibody and memory B cell responses in SARS-CoV-2 naïve and recovered individuals following mRNA vaccination.

Novel mRNA vaccines for SARS-CoV-2 have been authorized for emergency use. Despite their efficacy in clinical trials, data on mRNA vaccine-induced immune responses are mostly limited to serological analyses. Here, we interrogated antibody and antigen-specific memory B cells over time in 33 SARS-CoV-2 naïve and 11 SARS-CoV-2 recovered subjects. SARS-CoV-2 naïve individuals required both vaccine doses for optimal increases in antibodies, particularly for neutralizing titers against the B.1.351 variant. Memory B cells specific for full-length spike protein and the spike receptor binding domain (RBD) were also efficiently primed by mRNA vaccination and detectable in all SARS-CoV-2 naive subjects after the second vaccine dose, though the memory B cell response declined slightly with age. In SARS-CoV-2 recovered individuals, antibody and memory B cell responses were significantly boosted after the first vaccine dose; however, there was no increase in circulating antibodies, neutralizing titers, or antigen-specific memory B cells after the second dose. This robust boosting after the first vaccine dose strongly correlated with levels of pre-existing memory B cells in recovered individuals, identifying a key role for memory B cells in mounting recall responses to SARS-CoV-2 antigens. Together, our data demonstrated robust serological and cellular priming by mRNA vaccines and revealed distinct responses based on prior SARS-CoV-2 exposure, whereby COVID-19 recovered subjects may only require a single vaccine dose to achieve peak antibody and memory B cell responses. These findings also highlight the utility of defining cellular responses in addition to serologies and may inform SARS-CoV-2 vaccine distribution in a resource-limited setting.

Longitudinal Analysis Reveals Distinct Antibody and Memory B Cell Responses in SARS-CoV2 Naïve and Recovered Individuals Following mRNA Vaccination.

Novel mRNA vaccines for SARS-CoV2 have been authorized for emergency use and are currently being administered to millions of individuals worldwide. Despite their efficacy in clinical trials, there is limited data on vaccine-induced immune responses in individuals with a prior SARS-CoV2 infection compared to SARS-CoV2 naïve subjects. Moreover, how mRNA vaccines impact the development of antibodies as well as memory B cells in COVID-19 experienced versus COVID-19 naïve subjects remains poorly understood. In this study, we evaluated antibody responses and antigen-specific memory B cell responses over time in 33 SARS-CoV2 naïve and 11 SARS-CoV2 recovered subjects. mRNA vaccination induced significant antibody and memory B cell responses against full-length SARS-CoV2 spike protein and the spike receptor binding domain (RBD). SARS-CoV2 naïve individuals benefitted from both doses of mRNA vaccine with additional increases in antibodies and memory B cells following booster immunization. In contrast, SARS-CoV2 recovered individuals had a significant immune response after the first dose with no increase in circulating antibodies or antigen-specific memory B cells after the second dose. Moreover, the magnitude of the memory B cell response induced by vaccination was lower in older individuals, revealing an age-dependence to mRNA vaccine-induced B cell memory. Side effects also tended to associate with post-boost antibody levels, but not with post-boost memory B cells, suggesting that side effect severity may be a surrogate of short-term antibody responses. The frequency of pre-vaccine antigen-specific memory B cells in SARS-CoV2 recovered individuals strongly correlated with post-vaccine antibody levels, supporting a key role for memory B cells in humoral recall responses to SARS-CoV2. This observation may have relevance for future booster vaccines and for responses to viral variants that partially escape pre-existing antibodies and require new humoral responses to be generated from memory B cells. Finally, post-boost antibody levels were not correlated with post-boost memory responses in SARS-CoV2 naïve individuals, indicating that short-term antibody levels and memory B cells are complementary immunological endpoints that should be examined in tandem when evaluating vaccine response. Together, our data provide evidence of both serological response and immunological memory following mRNA vaccination that is distinct based on prior SARS-CoV2 exposure. These findings may inform vaccine distribution in a resource-limited setting.

Reduced social investigation and increased injurious behavior in transgenic 5xFAD mice.

Social withdrawal and agitation/aggression are common behavioral and psychological symptoms of dementia presented by Alzheimer's disease (AD) patients, with males exhibiting more aggressive behaviors than females. Some transgenic mouse models of AD also exhibit social withdrawal and aggression, but many of these models only recapitulate the early stages of the disease. By comparison, the 5xFAD mouse model of AD exhibits rapid, progressive neurodegeneration, and is suitable for modeling cognitive and behavioral deficits at early, mid-, and late-stage disease progression. Anecdotal reports suggest that transgenic 5xFAD males exhibit high levels of aggression compared to wild-type controls, but to date, indirect genetic effects in this strain have not been studied. We measured home-cage behaviors in 5xFAD males housed in three different group-housing conditions (transgenic-only, wild-type only, and mixed-genotype) and social approach behaviors when exposed to a novel free-roaming or restrained, wild-type or transgenic conspecific. Transgenic-only home cages required earlier separation due to injuries arising from aggression compared to wild-type-only or mixed-genotype cages, despite no obvious increase in the frequency of aggressive behaviors. Transgenic 5xFAD males and females also spent less time investigating free-roaming conspecifics compared to wild-type controls, but they showed normal investigation of restrained conspecifics; the genotype of the conspecific did not affect approach behavior, and there was no aggression observed in transgenic males. These findings provide evidence in an animal model that amyloid pathology ultimately leads to avoidance of novel social stimuli, and that frequent interactions between individuals exhibiting an AD phenotype further exacerbates aggressive behaviors.

Characterization of the vibrational properties of copper difluoride anion and neutral ground states via direct and indirect photodetachment spectroscopy.

Photoelectron spectra of 63CuF2- are reported at wavelengths 310 nm, 346.6 nm, and 350.1 nm, obtained via velocity map imaging. The photoelectron angular distributions allow for the unambiguous assignment of a 2Σg+ neutral CuF2 ground state. Vibrational analysis of the direct detachment transitions in the spectra enables accurate determination of the anion and neutral bond length difference (0.073 Å), adiabatic electron affinity of CuF2 (3.494 eV) and symmetric stretching (500 cm-1, anion, and 630 cm-1, neutral) and antisymmetric stretching (610 cm-1, anion, and 782 cm-1 neutral) frequencies of the ground electronic states. Strongly photon energy dependent intensities are also observed for select transitions. Equation-of-motion coupled-cluster singles and doubles calculations augmented by a complex absorbing potential reveal a metastable 1Πg anion state which is optically accessible due to Renner-Teller coupling. Mediation of the detachment process by this state allows measurement of the bending frequencies (177 cm-1, anion, and 200 cm-1, neutral) completing the inventory of experimentally measured vibrational properties of the ground electronic states.

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes.

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.