Animal tracing with sulfur isotopes: Spatial segregation and climate variability in Africa likely contribute to population trends of a migratory songbird.

Climatic conditions affect animals but range-wide impacts at the population level remain largely unknown, especially in migratory species. However, studying climate-population relationships is still challenging in small migrants due to a lack of efficient and cost-effective geographic tracking method. Spatial distribution patterns of environmental stable isotopes (so called 'isoscapes') generally overcome these limitations but none of the currently available isoscapes provide a substantial longitudinal gradient in species-rich sub-Saharan Africa. In this region, sulphur (δ34 S) has not been sufficiently explored on a larger scale. We developed a δ34 S isoscape to trace animal origins in sub-Saharan Africa by coupling known-origin samples from tracked migratory birds with continental remotely sensed environmental data building on environment-δ34 S relationships using a flexible machine learning technique. Furthermore, we link population-specific nonbreeding grounds with interannual climatic variation that might translate to breeding population trends. The predicted δ34 S isotopic map featured east-west and coast-to-inland isotopic gradients and was applied to predict nonbreeding grounds of three breeding populations of Eurasian Reed Warblers Acrocephalus scirpaceus with two distinct migratory phenotypes. Breeding populations as well as migratory phenotypes exhibited large-scale segregation within the African nonbreeding range. These regions also differed substantially in the interannual climatic variation, with higher interannual variability in the eastern part of the range during 2001-2012. Over the same period, the eastern European breeding population seemed to have experienced a more steep decline in population size. The link between migratory patterns and large-scale climatic variability appears important to better understand population trajectories in many declining migratory animals. We believe animal tracing using sulphur isotopes will facilitate these efforts and offers manifold ecological and forensic applications in the biodiversity hotspot of sub-Saharan Africa.

Culture Conditions for Human Induced Pluripotent Stem Cell-Derived Schwann Cells: A Two-Centre Study.

Adult human Schwann cells represent a relevant tool for studying peripheral neuropathies and developing regenerative therapies to treat nerve damage. Primary adult human Schwann cells are, however, difficult to obtain and challenging to propagate in culture. One potential solution is to generate Schwann cells from human induced pluripotent stem cells (hiPSCs). Previously published protocols, however, in our hands did not deliver sufficient viable cell numbers of hiPSC-derived Schwann cells (hiPSC-SCs). We present here, two modified protocols from two collaborating laboratories that overcome these challenges. With this, we also identified the relevant parameters to be specifically considered in any proposed differentiation protocol. Furthermore, we are, to our knowledge, the first to directly compare hiPSC-SCs to primary adult human Schwann cells using immunocytochemistry and RT-qPCR. We conclude the type of coating to be important during the differentiation process from Schwann cell precursor cells or immature Schwann cells to definitive Schwann cells, as well as the amounts of glucose in the specific differentiation medium to be crucial for increasing its efficiency and the final yield of viable hiPSC-SCs. Our hiPSC-SCs further displayed high similarity to primary adult human Schwann cells.

Safety of breast/chest-feeding by those infected by SARS-CoV-2.

PURPOSE OF REVIEW: One important question from the outset of the pandemic has been whether a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected person's milk might be a vehicle for SARS-CoV-2 transmission. This review summarizes the most recent data on this topic. RECENT FINDINGS: A SARS-CoV-2 sIgA response in milk after infection is very common. To date, there has been no evidence that SARS-CoV-2 transmits via human milk. Though viral RNA has been identified in a minority of milk samples studied, infectious virus particles have not. SUMMARY: The highly dominant transmission route for SARS-CoV-2 is via inhalation of respiratory droplets containing virus particles. Other routes of transmission are possible, including fecal-oral, trans-placental, and to a much lesser extent, via a contaminated surface. SARS-CoV-2 cannot transmit via human milk. There is no evidence that infants should be separated from SARS-CoV-2-infected mothers who are well enough to establish or continue breastfeeding.

Evaluation of phenytoin loading doses in overweight patients using actual versus adjusted body weight.

BACKGROUND AND OBJECTIVES: The appropriate loading dose strategy for phenytoin/fosphenytoin in overweight patients is unknown. A small pharmacokinetic study indicated that overweight patients have a higher volume of distribution and potentially would benefit from using adjusted body weight (AdjBW) instead of actual body weight (ABW) to calculate the loading dose. The purpose of this study was to determine the optimal loading dose strategy of phenytoin in patients whose ABW is greater than 120% of their ideal body weight (IBW) using either ABW or AdjBW for calculation of the loading dose. METHODS: This was a single center, retrospective study which included patients who received a loading dose of phenytoin of at least 10 mg/kg based on ABW, had a phenytoin level drawn <6 h after the end of the dose, and weighed ≥120% of their IBW. Patients were excluded if they received intramuscular phenytoin or fosphenytoin or were prescribed phenytoin prior to the loading dose. Patients were divided into two groups, those who were dosed using their AdjBW versus those dosed using ABW. The primary outcome was achievement of therapeutic phenytoin level of 10-20 mcg/mL. Secondary outcomes included achievement of a subtherapeutic or supratherapeutic level. RESULTS: A total of 195 patients (128 in AdjBW group and 67 in ABW group) met criteria for inclusion. Patients in the AdjBW group weighed more (96.2 kg vs. 91.2 kg, p = 0.04) and received a lower dose in milligrams (1364 vs. 1760, p < 0.0001) and in mg/kg of ABW (14.2 vs. 19.3, p < 0.0001). The primary outcome was achieved in 74% of patients in the AdjBW group and 57% of patients in the ABW group (p = 0.02). Patients in the ABW group were more likely to have a supratherapeutic level (43% vs. 22%, p = 0.003), although adverse reactions did not differ between the groups. DISCUSSION: Patients weighing >120% of their IBW (average body mass index 33.5 kg/m2) who received a 20 mg/kg loading dose based on AdjBW were more likely to achieve a therapeutic phenytoin concentration compared to those dosed based on ABW. Further research is needed to correlate this finding with clinical outcomes, such as resolution of status epilepticus.

Effect of Aß Oligomers on Neuronal APP Triggers a Vicious Cycle Leading to the Propagation of Synaptic Plasticity Alterations to Healthy Neurons.

Alterations of excitatory synaptic function are the strongest correlate to the pathologic disturbance of cognitive ability observed in the early stages of Alzheimer's disease (AD). This pathologic feature is driven by amyloid-ß oligomers (Aßos) and propagates from neuron to neuron. Here, we investigated the mechanism by which Aßos affect the function of synapses and how these alterations propagate to surrounding healthy neurons. We used complementary techniques ranging from electrophysiological recordings and molecular biology to confocal microscopy in primary cortical cultures, and from acute hippocampal and cortical slices from male wild-type and amyloid precursor protein (APP) knock-out (KO) mice to assess the effects of Aßos on glutamatergic transmission, synaptic plasticity, and dendritic spine structure. We showed that extracellular application of Aßos reduced glutamatergic synaptic transmission and long-term potentiation. These alterations were not observed in APP KO neurons, suggesting that APP expression is required. We demonstrated that Aßos/APP interaction increases the amyloidogenic processing of APP leading to intracellular accumulation of newly produced Aßos. Intracellular Aßos participate in synaptic dysfunctions as shown by pharmacological inhibition of APP processing or by intraneuronal infusion of an antibody raised against Aßos. Furthermore, we provide evidence that following APP processing, extracellular release of Aßos mediates the propagation of the synaptic pathology characterized by a decreased spine density of neighboring healthy neurons in an APP-dependent manner. Together, our data unveil a complementary role for Aßos in AD, while intracellular Aßos alter synaptic function, extracellular Aßos promote a vicious cycle that propagates synaptic pathology from diseased to healthy neurons.SIGNIFICANCE STATEMENT Here we provide the proof that a vicious cycle between extracellular and intracellular pools of Aß oligomers (Aßos) is required for the spreading of Alzheimer's disease (AD) pathology. We showed that extracellular Aßos propagate excitatory synaptic alterations by promoting amyloid precursor protein (APP) processing. Our results also suggest that subsequent to APP cleavage two pools of Aßos are produced. One pool accumulates inside the cytosol, inducing the loss of synaptic plasticity potential. The other pool is released into the extracellular space and contributes to the propagation of the pathology from diseased to healthy neurons. Pharmacological strategies targeting the proteolytic cleavage of APP disrupt the relationship between extracellular and intracellular Aß, providing a therapeutic approach for the disease.

An HIV Vaccine Targeting the V2 Region of the HIV Envelope Induces a Highly Durable Polyfunctional Fc-Mediated Antibody Response in Rhesus Macaques.

The HIV vaccine field now recognizes the potential importance of generating polyfunctional antibodies (Abs). The only clinical HIV vaccine trial to date to show significant efficacy (RV144) found that reduced infection rates correlated with the level of nonneutralizing Abs specific for the V2 region of the envelope glycoprotein. We have conducted a comprehensive preclinical reverse vaccinology-based vaccine program that has included the design and production and testing of numerous scaffolded V2 region immunogens. The most immunogenic vaccine regimen in nonhuman primates among those studied as part of this program consisted of a cocktail of three immunogens presenting V2 from different viruses and clades in the context of different scaffolds. Presently we demonstrate that the V2-specific Ab response from this regimen was highly durable and functionally diverse for the duration of the study (25 weeks after the final immunization). The total IgG binding response at this late time point exhibited only an ∼5× reduction in potency. Three immunizations appeared essential for the elicitation of a strong Ab-dependent cellular cytotoxicity (ADCC) response for all animals, as opposed to the Ab-dependent cellular phagocytosis (ADCP) and virus capture responses, which were comparably potent after only 2 immunizations. All functionalities measured were highly durable through the study period. Therefore, testing this vaccine candidate for its protective capacity is warranted.IMPORTANCE The only HIV vaccine trial for which protective efficacy was detected correlated this efficacy with V2-specific Abs that were effectively nonneutralizing. This result has fueled a decade of HIV vaccine research focused on designing an HIV vaccine capable of eliciting V2-focused, polyfunctional Abs that effectively bind HIV and trigger various leukocytes to kill the virus and restrict viral spread. From the numerous vaccine candidates designed and tested as part of our V2-focused preclinical vaccine program, we have identified immunogens and a vaccine regimen that induces a highly durable and polyfunctional V2-focused Ab response in rhesus macaques, described herein.

IFITM proteins drive type 2 T helper cell differentiation and exacerbate allergic airway inflammation.

The interferon-inducible transmembrane (Ifitm/Fragilis) genes encode homologous proteins that are induced by IFNs. Here, we show that IFITM proteins regulate murine CD4+ Th cell differentiation. Ifitm2 and Ifitm3 are expressed in wild-type (WT) CD4+ T cells. On activation, Ifitm3 was downregulated and Ifitm2 was upregulated. Resting Ifitm-family-deficient CD4+ T cells had higher expression of Th1-associated genes than WT and purified naive Ifitm-family-deficient CD4+ T cells differentiated more efficiently to Th1, whereas Th2 differentiation was inhibited. Ifitm-family-deficient mice, but not Ifitm3-deficient mice, were less susceptible than WT to induction of allergic airways disease, with a weaker Th2 response and less severe disease and lower Il4 but higher Ifng expression and IL-27 secretion. Thus, the Ifitm family is important in adaptive immunity, influencing Th1/Th2 polarization, and Th2 immunopathology.

Reduced Cell-Associated DNA and Improved Viral Control in Macaques following Passive Transfer of a Single Anti-V2 Monoclonal Antibody and Repeated Simian/Human Immunodeficiency Virus Challenges.

A high level of V1V2-specific IgG antibodies (Abs) in vaccinees' sera was the only independent variable that correlated with a reduced risk of human immunodeficiency virus (HIV) acquisition in the RV144 clinical trial. In contrast, IgG avidity, antibody neutralization, and antibody-dependent cellular cytotoxicity each failed as independent correlates of infection. Extended analyses of RV144 samples demonstrated the antiviral activities of V1V2-specific vaccine-induced antibodies. V2-specific antibodies have also been associated with protection from simian immunodeficiency virus (SIV), and the V2i-specific subset of human monoclonal antibodies (MAbs), while poor neutralizers, mediates Fc-dependent antiviral functions in vitro The objective of this study was to determine the protective efficacy of a V2i-specific human MAb, 830A, against mucosal simian/human immunodeficiency virus (SHIV) challenge. V2i MAb binding sites overlap the integrin binding site in the V2 region and are similar to the epitopes bound by antibodies associated with reduced HIV infection rates in RV144. Because the IgG3 subclass was a correlate of reduced infection rates in RV144, we compared passive protection by both IgG1 and IgG3 subclasses of V2i MAb 830A. This experiment represents the first in vivo test of the hypothesis emanating from RV144 and SIV studies that V2i Abs can reduce the risk of infection. The results show that passive transfer with a single V2i MAb, IgG1 830A, reduced plasma and peripheral blood mononuclear cell (PBMC) virus levels and decreased viral DNA in lymphoid tissues compared to controls, but too few animals remained uninfected to achieve significance in reducing the risk of infection. Based on these findings, we conclude that V2i antibodies can impede virus seeding following mucosal challenge, resulting in improved virus control.IMPORTANCE Since the results of the HIV RV144 clinical trial were reported, there has been significant interest in understanding how protection was mediated. Antibodies directed to a subregion of the envelope protein called V1V2 were directly correlated with a reduced risk, and surprisingly low virus neutralization was observed. To determine whether these antibodies alone could mediate protection, we used a human monoclonal antibody directed to V2 with properties similar to those elicited in the vaccine trial for passive infusions in rhesus macaques and challenge with SHIV. The single V2 antibody at the dose given did not significantly reduce the number of infections, but there was a significant reduction in the seeding of virus to the lymph nodes and a decrease in plasma viremia in the HIV antibody-infused macaques compared with the control antibody-infused animals. This finding shows that V2 antibodies mediate antiviral activities in vivo that could contribute to a protective HIV vaccine.

Plasticity and Epitope Exposure of the HIV-1 Envelope Trimer.

We recently showed that mutations in the HIV-1 envelope (Env) destabilize the V3 loop, rendering neutralization-resistant viruses sensitive to V3-directed monoclonal antibodies (MAbs). Here, we investigated the propagation of this effect on other Env epitopes, with special emphasis on V2 loop exposure. Wild-type JR-FL and 19 mutant JR-FL pseudoviruses were tested for neutralization sensitivity to 21 MAbs specific for epitopes in V2, the CD4 binding site (CD4bs), and the CD4-induced (CD4i) region. Certain glycan mutants, mutations in the gp120 hydrophobic core, and mutations in residues involved in intraprotomer interactions exposed epitopes in the V2i region (which overlies the α4ß7 integrin binding site) and the V3 crown, suggesting general destabilization of the distal region of the trimer apex. In contrast, other glycan mutants, mutations affecting interprotomer interactions, and mutations affecting the CD4bs exposed V3 but not V2i epitopes. These data indicate for the first time that V3 can move independently of V2, with V3 pivoting out from its "tucked" position in the trimer while apparently leaving the V2 apex intact. Notably, none of the mutations exposed V2 epitopes without also exposing V3, suggesting that movement of V2 releases V3. Most mutations increased sensitivity to CD4bs-directed MAbs without exposure of the CD4i epitope, implying these mutations facilitate the trimers' maintenance of an intermediate energy state between open and closed conformations. Taken together, these data indicate that several transient Env epitopes can be rendered more accessible to antibodies (Abs) via specific mutations, and this may facilitate the design of V1V2-targeting immunogens.IMPORTANCE Many epitopes of the HIV envelope (Env) spike are relatively inaccessible to antibodies (Abs) compared to their exposure in the open Env conformation induced by receptor binding. However, the reduced infection rate that resulted from the vaccine used in the RV144 HIV-1 vaccine trial was correlated with the elicitation of V2- and V3-directed antibodies. Previously, we identified various mechanisms responsible for destabilizing the V3 loop; here, we determined, via mutation of numerous Env residues, which of these elements maintain the V1V2 loop in an inaccessible state and which expose V1V2 and/or V3 epitopes. Notably, our data indicate that V3 can move independently of V2, but none of the mutations studied expose V2 epitopes without also exposing V3. Additionally, V1V2 can be rendered more accessible to Abs via specific mutations, facilitating the development of engineered V2 immunogens.

Rationally Designed Vaccines Targeting the V2 Region of HIV-1 gp120 Induce a Focused, Cross-Clade-Reactive, Biologically Functional Antibody Response.

Strong antibody (Ab) responses against V1V2 epitopes of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope (Env) correlated with reduced infection rates in studies of HIV, simian-human immunodeficiency virus (SHIV), and simian immunodeficiency virus (SIV). In order to focus the Ab response on V1V2, we used six V1V2 sequences and nine scaffold proteins to construct immunogens which were tested using various immunization regimens for their ability to induce cross-reactive and biologically active V2 Abs in rabbits. A prime/boost immunization strategy was employed using gp120 DNA and various V1V2-scaffold proteins. The rabbit polyclonal Ab responses (i) were successfully focused on the V1V2 region, with weak or only transient responses to other Env epitopes, (ii) displayed broad cross-reactive binding activity with gp120s and the V1V2 regions of diverse strains from clades B, C, and E, (iii) included V2 Abs with specificities similar to those found in HIV-infected individuals, and (iv) remained detectable ≥1 year after the last boosting dose. Importantly, sera from rabbits receiving V1V2-scaffold immunogens displayed Ab-dependent cellular phagocytosis whereas sera from rabbits receiving only gp120 did not. The results represent the first fully successful example of reverse vaccinology in the HIV vaccine field with rationally designed epitope scaffold immunogens inducing Abs that recapitulate the epitope specificity and biologic activity of the human monoclonal Abs from which the immunogens were designed. Moreover, this is the first immunogenicity study using epitope-targeting, rationally designed vaccine constructs that induced an Fc-mediated activity associated with protection from infection with HIV, SIV, and SHIV. IMPORTANCE: Novel immunogens were designed to focus the antibody response of rabbits on the V1V2 epitopes of HIV-1 gp120 since such antibodies were associated with reduced infection rates of HIV, SIV, and SHIV. The vaccine-induced antibodies were broadly cross-reactive with the V1V2 regions of HIV subtypes B, C and E and, importantly, facilitated Fc-mediated phagocytosis, an activity not induced upon immunization of rabbits with gp120. This is the first immunogenicity study of vaccine constructs that focuses the antibody response on V1V2 and induces V2-specific antibodies with the ability to mediate phagocytosis, an activity that has been associated with protection from infection with HIV, SIV, and SHIV.

A developmentally plastic adult mouse kidney cell line spontaneously generates multiple adult kidney structures.

Despite exciting new possibilities for regenerative therapy posed by the ability to induce pluripotent stem cells, recapitulation of three-dimensional kidneys for repair or replacement has not been possible. ARID3a-deficient mouse tissues generated multipotent, developmentally plastic cells. Therefore, we assessed the adult mouse ARID3a-/- kidney cell line, KKPS5, which expresses renal progenitor surface markers as an alternative cell source for modeling kidney development. Remarkably, these cells spontaneously developed into multicellular nephron-like structures in vitro, and engrafted into immunocompromised medaka mesonephros, where they formed mouse nephron structures. These data implicate KKPS5 cells as a new model system for studying kidney development.

Identification of an HIV-1 clade A envelope that exhibits broad antigenicity and neutralization sensitivity and elicits antibodies targeting three distinct epitopes.

Broadly neutralizing antibodies (bNAbs) PG9 and PG16 were isolated from an International AIDS Vaccine Initiative (IAVI) Protocol G subject infected with human immunodeficiency virus type 1 (HIV-1) clade A. Both antibodies are highly potent and neutralize greater than 70% of viruses tested. We sought to begin immunogen design based on viral sequences from this patient; however, pseudoviruses prepared with 19 envelope sequences from this subject were resistant to neutralization by PG9 and PG16. Therefore, we used a bioinformatics approach to identify closely related viruses that were potentially sensitive to PG9 and PG16. A most-recent common ancestor (MRCA) sequence for the viral envelope (Env) was determined and aligned with 99 subtype A gp160 sequences from the Los Alamos HIV database. Virus BG505.W6M.ENV.C2 (BG505) was found to have the highest degree of homology (73%) to the MRCA sequence. Pseudoviruses prepared with this Env were sensitive to neutralization with a broad panel of bNAbs, including PG9 and PG16. When expressed by 293T cells as soluble gp120, the BG505 monomer bound well to both PG9 and PG16. We further showed that a point mutation (L111A) enabled more efficient production of a stable gp120 monomer that preserves the major neutralization epitopes. Finally, we showed that an adjuvanted formulation of this gp120 protein elicited neutralizing antibodies in rabbits (following a gp120 DNA vaccine prime) and that the antisera competed with bNAbs from 3 classes of nonoverlapping epitopes. Thus, the BG505 Env protein warrants further investigation as an HIV vaccine candidate, as a stand-alone protein, or as a component of a vaccine vector.

In vivo antiviral efficacy of LCTG-002, a pooled, purified human milk secretory IgA product, against SARS-CoV-2 in a murine model of COVID-19.

Immunoglobulin A (IgA) is the most abundant antibody (Ab) in human mucosae, with secretory form (sIgA) being dominant and uniquely stable. sIgA is challenging to produce recombinantly but is naturally found in human milk, which could be considered a global resource for this biologic, justifying its development as a mucosal therapeutic. Presently, SARS-CoV-2 was utilized as a model mucosal pathogen, and methods were developed to efficiently extract human milk sIgA from donors who were naïve to SARS-CoV-2 or had recovered from infection that elicited high-titer anti-SARS-CoV-2 Spike sIgA in their milk (pooled to make LCTG-002). Mass spectrometry determined that proteins with a relative abundance of 1% or greater were all associated with sIgA. Western blot demonstrated that all batches consisted predominantly of sIgA. Compared to control IgA, LCTG-002 demonstrated significantly higher Spike binding (mean endpoint of 0.87 versus 5.87). LCTG-002 was capable of blocking the Spike receptor-binding domain - angiotensin-converting enzyme 2 (ACE2) interaction with significantly greater potency compared to control (mean LCTG-002 IC50 154ug/mL versus 50% inhibition not achieved for control), and exhibited significant neutralization activity against Spike-pseudotyped virus infection (mean LCTG-002 IC50 49.8ug/mL versus 114.5ug/mL for control). LCTG-002 was tested for its capacity to reduce viral lung burden in K18+hACE2 transgenic mice inoculated with SARS-CoV-2. LCTG-002 significantly reduced SARS-CoV-2 titers compared to control when administered at 0.25 mg/day or 1 mg/day, with a maximum TCID50 reduction of 4.9 logs. This innovative study demonstrates that LCTG-002 is highly pure and efficacious in vivo, supporting further development of milk-derived, polyclonal sIgA therapeutics.

Medaka fish, Oryzias latipes, as a model for human obesity-related glomerulopathy.

Obesity, an ongoing significant public health problem, is a part of complex disease characterized as metabolic syndrome. Medaka and zebrafish are useful aquatic experimental animals widely used in the field of toxicology and environmental health sciences and as a human disease models. In medaka, simple feeding of a high fat diet (HFD) can induce body weight gain, excessive accumulation of visceral adipose tissue, hyperglycemia, hyperlipidemia, and steatohepatitis, which mimics human metabolic syndrome. In the present study, to explore the possibility that the adult medaka fed with HFD (HFD-medaka) can be used as an animal model for human metabolic syndrome-associated glomerular disease, including obesity-related glomerulopathy (ORG), we analyzed structural alterations and protein expression in the mesonephric kidney of HFD-medaka. We found that the histopathology was consistent with glomerulomegaly accompanied by the dilation of glomerular capillaries and proliferative expansion of the mesangium, a condition partially comparable to human ORG. Moreover, expressions of several kinds of kidney disease-related proteins (such as MYH9, SM22α) were significantly elevated. Thus, the HFD-medaka has a high potential as an animal model useful for exploring the mechanism underling human ORG.

Structural disorganization of pronephric glomerulus in zebrafish mpp5a/nagie oko mutant.

BACKGROUND: The podocyte slit diaphragm (SD) is an essential component of the selective filtration barrier in the glomerulus. Several structural proteins required for formation and maintenance of SD have been identified; however, molecular mechanisms regulating these proteins are still limited. RESULTS: Here, we demonstrate that MAGUK p55 subfamily member 5a (Mpp5a)/Nagie oko, a component of the Crb multi-protein complex, was colocalized with an SD-associated protein ZO-1 in the zebrafish pronephric glomerulus. To characterize the function of Mpp5a, zebrafish mpp5a(m520) mutant embryos, which are known to have defects in cardiac and neuronal morphogenesis, were analyzed. These mutants failed to merge the bilateral glomerular primordia and to form the glomerular capillary and mesangium, but the foot processes and SD showed normal appearance. The structural disorganization in the mpp5a(m520) mutant glomerulus was quite similar to that of a cardiac troponin T2a/tnnt2a/silent heart knockdown zebrafish, which exhibited circulatory failure due to lack of heart beating. CONCLUSIONS: Mpp5a is not prerequisite to form podocyte slit diaphragm in the pronephric glomerular development in zebrafish. The structural disorganization of the pronephric glomerulus in the mpp5a(m520) mutant is likely to result from circulatory failure, rather than the anomaly of Mpp5a protein in the glomerulus.

The Secretory IgA Response in Human Milk Against the SARS-CoV-2 Spike Is Highly Durable and Neutralizing for At Least 1 Year of Lactation Postinfection.

Background: Although in the early pandemic period COVID-19 pathology among young children and infants was typically less severe compared with that observed among adults, this has not remained entirely consistent as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have emerged. There is an enormous body of evidence demonstrating the benefits of human milk antibodies (Abs) in protecting infants against a wide range of enteric and respiratory infections. It is highly plausible that the same holds true for protection against SARS-CoV-2 as this virus infects cells of the gastrointestinal and respiratory mucosae. Understanding the durability of a human milk Ab response over time after infection is critical. Objective: Previously, we examined the Abs present in milk of those recently infected with SARS-CoV-2 and concluded that the response was secretory immunoglobulin A (sIgA) dominant and that these titers were highly correlated with neutralization potency. The present study aimed to monitor the durability of the SARS-CoV-2 IgA and secretory Ab (sAb) response in milk from COVID-19-recovered lactating individuals over 12 months in the absence of vaccination or reinfection. Results: This analysis revealed a robust and durable spike-specific milk sIgA response, and at 9-12 months after infection, 88% of the samples exhibited titers above the positive cutoff for IgA and 94% were above the cutoff for sAb. Fifty percent of participants exhibited less than twofold reduction of spike-specific IgA through 12 months. A strong, significant positive correlation between IgA and sAb against spike persisted throughout the study period. Nucleocapsid-specific Abs were also assessed, which revealed significant background or cross-reactivity of milk IgA against this immunogen, as well as limited/inconsistent durability compared with Spike titers. Conclusion: These data suggest that lactating individuals are likely to continue producing spike-specific Abs in their milk for 1 year or more, which may provide critical passive immunity to infants against SARS-CoV-2 throughout the lactation period.

Assessment of human milk samples obtained pre and post-influenza vaccination reveals a poor boosting of seasonally-relevant, hemagglutinin-specific antibodies.

Introduction: Influenza (flu) vaccination prevented over 100,000 hospitalizations and 7000 deaths from flu over the 2019-2020 season in the USA. Infants <6 months are the most likely to die from flu, though flu vaccines are only licensed for infants >6 months old. Therefore, it is recommended that flu vaccination occur during pregnancy, as this reduces severe complications; however, vaccination rates are suboptimal, and vaccination is also recommended postpartum. For breast/chest-fed infants, the vaccine is believed to elicit protective and robust seasonally-specific milk antibody (Ab). Few comprehensive studies exist examining Ab responses in milk after vaccination, with none measuring secretory Ab (sAb). Determining whether sAbs are elicited is critical, as this Ab class is highly stable in milk and mucosae. Methods: In the present study, our aim was to determine to what extent specific Ab titers in the milk of lactating people were boosted after seasonal influenza vaccination. Over the 2019-2020 and 2020-2021 seasons, milk was obtained pre- and post-vaccination and assessed for specific IgA, IgG, and sAb against relevant hemagglutinin (HA) antigens by a Luminex immunoassay. Results: IgA and sAb were not found to be significantly boosted, while only IgG titers against B/Phuket/3073/2013, included in vaccines since 2015, exhibited an increase. Across the 7 immunogens examined, as many as 54% of samples exhibited no sAb boost. No significant differences for IgA, sAb, or IgG boosting were measured between seasonally-matched versus mismatched milk groups, indicating boosting was not seasonally-specific. No correlations between IgA and sAb increases were found for 6/8 HA antigens. No boost in IgG- or IgA-mediated neutralization post vaccination was observed. Discussion: This study highlights the critical need to redesign influenza vaccines with the lactating population in mind, wherein the aim should be to elicit a potent seasonally-specific sAb response in milk. As such, this population must be included in clinical studies.

The secretory IgA (sIgA) response in human milk against the SARS-CoV-2 Spike is highly durable and neutralizing for at least 1 year of lactation post-infection.

Although in the early pandemic period, COVID-19 pathology among young children and infants was typically less severe compared to that observed among adults, this has not remained entirely consistent as SARS-CoV-2 variants have emerged. There is an enormous body of evidence demonstrating the benefits of human milk antibodies (Abs) in protecting infants against a wide range of enteric and respiratory infections. It is highly plausible that the same holds true for protection against SARS-CoV-2, as this virus infects cells of the gastrointestinal and respiratory mucosae. Understanding the durability of a human milk Ab response over time after infection is critical. Previously, we examined the Abs present in milk of those recently infected with SARS-CoV-2, and concluded that the response was secretory IgA (sIgA)-dominant and that these titers were highly correlated with neutralization potency. The present study aimed to monitor the durability of the SARS-CoV-2 IgA and secretory Ab (sAb) response in milk from COVID-19-recovered lactating individuals over 12 months, in the absence of vaccination or re-infection. This analysis revealed a robust and durable Spike-specific milk sIgA response, that at 9-12 months after infection, 88% of the samples exhibited titers above the positive cutoff for IgA and 94% were above cutoff for sAb. Fifty percent of participants exhibited less than a 2-fold reduction of Spike-specific IgA through 12 months. A strong significant positive correlation between IgA and sAb against Spike persisted throughout the study period. Nucleocapsid-specific Abs were also assessed, which revealed significant background or cross reactivity of milk IgA against this immunogen, as well as limited/inconsistent durability compared to Spike titers. These data suggests that lactating individuals are likely to continue producing Spike-specific Abs in their milk for 1 year or more, which may provide critical passive immunity to infants against SARS-CoV-2 throughout the lactation period.

In Vivo Antiviral Efficacy of LCTG-002, a Pooled, Purified Human Milk Secretory IgA product, Against SARS-CoV-2 in a Murine Model of COVID-19.

Immunoglobulin A (IgA) is the most abundant antibody (Ab) in human mucosal compartments including the respiratory tract, with the secretory form of IgA (sIgA) being dominant and uniquely stable in these environments. sIgA is naturally found in human milk, which could be considered a global resource for this biologic, justifying the development of human milk sIgA as a dedicated airway therapeutic for respiratory infections such as SARS-CoV-2. In the present study, methods were therefore developed to efficiently extract human milk sIgA from donors who were either immunologically naïve to SARS-CoV-2 (pooled as a control IgA) or had recovered from a PCR-confirmed SARS-CoV-2 infection that elicited high-titer anti-SARS-CoV-2 Spike sIgA Abs in their milk (pooled together to make LCTG-002). Mass spectrometry determined that proteins with a relative abundance of 1.0% or greater were all associated with sIgA. None of the proteins exhibited statistically significant differences between batches. Western blot demonstrated all batches consisted predominantly of sIgA. Compared to control IgA, LCTG-002 demonstrated significantly higher binding to Spike, and was also capable of blocking the Spike - ACE2 interaction in vitro with 6.3x greater potency compared to control IgA (58% inhibition at ∼240ug/mL). LCTG-002 was then tested in vivo for its capacity to reduce viral burden in the lungs of K18+hACE2 transgenic mice inoculated with SARS-CoV-2. LCTG-002 was demonstrated to significantly reduce SARS-CoV-2 titers in the lungs compared to control IgA when administered at either 250ug/day or 1 mg/day, as measured by TCID50, plaque forming units (PFU), and qRT-PCR, with a maximum reduction of 4.9 logs. This innovative study demonstrates that LCTG-002 is highly pure, efficacious, and well tolerated in vivo, supporting further development of milk-derived, polyclonal sIgA therapeutics against SARS-CoV-2 and other mucosal infections.