Exposure of progressive immune dysfunction by SARS-CoV-2 mRNA vaccination in patients with chronic lymphocytic leukemia: A prospective cohort study.

BACKGROUND: Patients with chronic lymphocytic leukemia (CLL) have reduced seroconversion rates and lower binding antibody (Ab) and neutralizing antibody (NAb) titers than healthy individuals following Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) mRNA vaccination. Here, we dissected vaccine-mediated humoral and cellular responses to understand the mechanisms underlying CLL-induced immune dysfunction. METHODS AND FINDINGS: We performed a prospective observational study in SARS-CoV-2 infection-naïve CLL patients (n = 95) and healthy controls (n = 30) who were vaccinated between December 2020 and June 2021. Sixty-one CLL patients and 27 healthy controls received 2 doses of the Pfizer-BioNTech BNT162b2 vaccine, while 34 CLL patients and 3 healthy controls received 2 doses of the Moderna mRNA-1273 vaccine. The median time to analysis was 38 days (IQR, 27 to 83) for CLL patients and 36 days (IQR, 28 to 57) for healthy controls. Testing plasma samples for SARS-CoV-2 anti-spike and receptor-binding domain Abs by enzyme-linked immunosorbent assay (ELISA), we found that all healthy controls seroconverted to both antigens, while CLL patients had lower response rates (68% and 54%) as well as lower median titers (23-fold and 30-fold; both p < 0.001). Similarly, NAb responses against the then prevalent D614G and Delta SARS-CoV-2 variants were detected in 97% and 93% of controls, respectively, but in only 42% and 38% of CLL patients, who also exhibited >23-fold and >17-fold lower median NAb titers (both p < 0.001). Interestingly, 26% of CLL patients failed to develop NAbs but had high-titer binding Abs that preferentially reacted with the S2 subunit of the SARS-CoV-2 spike. Since these patients were also seropositive for endemic human coronaviruses (HCoVs), these responses likely reflect cross-reactive HCoV Abs rather than vaccine-induced de novo responses. CLL disease status, advanced Rai stage (III-IV), elevated serum beta-2 microglobulin levels (ß2m >2.4 mg/L), prior therapy, anti-CD20 immunotherapy (<12 months), and intravenous immunoglobulin (IVIg) prophylaxis were all predictive of an inability to mount SARS-CoV-2 NAbs (all p ≤ 0.03). T cell response rates determined for a subset of participants were 2.8-fold lower for CLL patients compared to healthy controls (0.05, 95% CI 0.01 to 0.27, p < 0.001), with reduced intracellular IFNγ staining (p = 0.03) and effector polyfunctionality (p < 0.001) observed in CD4+ but not in CD8+ T cells. Surprisingly, in treatment-naïve CLL patients, BNT162b2 vaccination was identified as an independent negative risk factor for NAb generation (5.8, 95% CI 1.6 to 27, p = 0.006). CLL patients who received mRNA-1273 had 12-fold higher (p < 0.001) NAb titers and 1.7-fold higher (6.5, 95% CI 1.3 to 32, p = 0.02) response rates than BNT162b2 vaccinees despite similar disease characteristics. The absence of detectable NAbs in CLL patients was associated with reduced naïve CD4+ T cells (p = 0.03) and increased CD8+ effector memory T cells (p = 0.006). Limitations of the study were that not all participants were subjected to the same immune analyses and that pre-vaccination samples were not available. CONCLUSIONS: CLL pathogenesis is characterized by a progressive loss of adaptive immune functions, including in most treatment-naïve patients, with preexisting memory being preserved longer than the capacity to mount responses to new antigens. In addition, higher NAb titers and response rates identify mRNA-1273 as a superior vaccine for CLL patients.

SARS-CoV-2 mRNA vaccination exposes progressive adaptive immune dysfunction in patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) patients have lower seroconversion rates and antibody titers following SARS-CoV-2 vaccination, but the reasons for this diminished response are poorly understood. Here, we studied humoral and cellular responses in 95 CLL patients and 30 healthy controls after two BNT162b2 or mRNA-2173 mRNA immunizations. We found that 42% of CLL vaccinees developed SARS-CoV-2-specific binding and neutralizing antibodies (NAbs), while 32% had no response. Interestingly, 26% were seropositive, but had no detectable NAbs, suggesting the maintenance of pre-existing endemic human coronavirus-specific antibodies that cross-react with the S2 domain of the SARS-CoV-2 spike. These individuals had more advanced disease. In treatment-naïve CLL patients, mRNA-2173 induced 12-fold higher NAb titers and 1.7-fold higher response rates than BNT162b2. These data reveal a graded loss of immune function, with pre-existing memory being preserved longer than the capacity to respond to new antigens, and identify mRNA-2173 as a superior vaccine for CLL patients.

Predictors of Nonseroconversion after SARS-CoV-2 Infection.

Not all persons recovering from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection develop SARS-CoV-2-specific antibodies. We show that nonseroconversion is associated with younger age and higher reverse transcription PCR cycle threshold values and identify SARS-CoV-2 viral loads in the nasopharynx as a major correlate of the systemic antibody response.

Antibody Responses to SARS-CoV-2 mRNA Vaccines Are Detectable in Saliva.

The approved Pfizer and Moderna mRNA vaccines are well known to induce serum antibody responses to the SARS-CoV-2 Spike (S)-protein. However, their abilities to elicit mucosal immune responses have not been reported. Saliva antibodies represent mucosal responses that may be relevant to how mRNA vaccines prevent oral and nasal SARS-CoV-2 transmission. Here, we describe the outcome of a cross-sectional study on a healthcare worker cohort (WELCOME-NYPH), in which we assessed whether IgM, IgG, and IgA antibodies to the S-protein and its receptor-binding domain (RBD) were present in serum and saliva samples. Anti-S-protein IgG was detected in 14/31 and 66/66 of saliva samples from uninfected participants after vaccine doses-1 and -2, respectively. IgA antibodies to the S-protein were present in 40/66 saliva samples after dose 2. Anti-S-protein IgG was present in every serum sample from recipients of 2 vaccine doses. Vaccine-induced antibodies against the RBD were also frequently present in saliva and sera. These findings may help our understanding of whether and how vaccines may impede SARS-CoV-2 transmission, including to oral cavity target cells.

Enhancing glycan occupancy of soluble HIV-1 envelope trimers to mimic the native viral spike.

Artificial glycan holes on recombinant Env-based vaccines occur when a potential N-linked glycosylation site (PNGS) is under-occupied, but not on their viral counterparts. Native-like SOSIP trimers, including clinical candidates, contain such holes in the glycan shield that induce strain-specific neutralizing antibodies (NAbs) or non-NAbs. To eliminate glycan holes and mimic the glycosylation of native BG505 Env, we replace all 12 NxS sequons on BG505 SOSIP with NxT. All PNGS, except N133 and N160, are nearly fully occupied. Occupancy of the N133 site is increased by changing N133 to NxS, whereas occupancy of the N160 site is restored by reverting the nearby N156 sequon to NxS. Hence, PNGS in close proximity, such as in the N133-N137 and N156-N160 pairs, affect each other's occupancy. We further apply this approach to improve the occupancy of several Env strains. Increasing glycan occupancy should reduce off-target immune responses to vaccine antigens.

Antibody responses to SARS-CoV-2 mRNA vaccines are detectable in saliva.

Vaccines are critical for curtailing the COVID-19 pandemic (1, 2). In the USA, two highly protective mRNA vaccines are available: BNT162b2 from Pfizer/BioNTech and mRNA-1273 from Moderna (3, 4). These vaccines induce antibodies to the SARS-CoV-2 S-protein, including neutralizing antibodies (NAbs) predominantly directed against the Receptor Binding Domain (RBD) (1-4). Serum NAbs are induced at modest levels within ~1 week of the first dose, but their titers are strongly boosted by a second dose at 3 (BNT162b2) or 4 weeks (mRNA-1273) (3, 4). SARS-CoV-2 is most commonly transmitted nasally or orally and infects cells in the mucosae of the respiratory and to some extent also the gastrointestinal tract (5). Although serum NAbs may be a correlate of protection against COVID-19, mucosal antibodies might directly prevent or limit virus acquisition by the nasal, oral and conjunctival routes (5). Whether the mRNA vaccines induce mucosal immunity has not been studied. Here, we report that antibodies to the S-protein and its RBD are present in saliva samples from mRNA-vaccinated healthcare workers (HCW). Within 1-2 weeks after their second dose, 37/37 and 8/8 recipients of the Pfizer and Moderna vaccines, respectively, had S-protein IgG antibodies in their saliva, while IgA was detected in a substantial proportion. These observations may be relevant to vaccine-mediated protection from SARS-CoV-2 infection and disease.

Complete Genome Sequence of Streptomyces Siphophage Sitrop.

Streptomyces sp. strain Mg1 is a Gram-positive soil bacterium capable of causing cell lysis and degradation of Bacillus subtilis colonies. Here, we report the 48,481-bp genome of Streptomyces sp. Mg1 siphophage Sitrop. With 77 predicted protein-coding genes and one tRNA, Sitrop shares 77% nucleotide sequence identity with the Streptomyces phage Verse.

Colorectal resection in emergency general surgery: An EAST multicenter trial.

OBJECTIVE: Evidence comparing stoma creation (STM) versus anastomosis after urgent or emergent colorectal resection is limited. This study examined outcomes after colorectal resection in emergency general surgery patients. METHODS: This was an Eastern Association for the Surgery of Trauma-sponsored prospective observational multicenter study of patients undergoing urgent/emergent colorectal resection. Twenty-one centers enrolled patients for 11 months. Preoperative, intraoperative, and postoperative variables were recorded. χ, Mann-Whitney U test, and multivariable logistic regression models were used to describe outcomes and risk factors for surgical complication/mortality. RESULTS: A total of 439 patients were enrolled (ANST, 184; STM, 255). The median (interquartile range) age was 62 (53-71) years, and the median Charlson Comorbidity Index (CCI) was 4 (1-6). The most common indication for surgery was diverticulitis (28%). Stoma group was older (64 vs. 58 years, p < 0.001), had a higher CCI, and were more likely to be immunosuppressed. Preoperatively, STM patients were more likely to be intubated (57 vs. 15, p < 0.001), on vasopressors (61 vs. 13, p < 0.001), have pneumoperitoneum (131 vs. 41, p < 0.001) or fecal contamination (114 vs. 33, p < 0.001), and had a higher incidence of elevated lactate (149 vs. 67, p < 0.001). Overall mortality was 13%, which was higher in STM patients (18% vs. 8%, p = 0.02). Surgical complications were more common in STM patients (35% vs. 25%, p = 0.02). On multivariable analysis, management with an open abdomen, intraoperative blood transfusion, and larger hospital size were associated with development of a surgical complication, while CCI, preoperative vasopressor use, steroid use, open abdomen, and intraoperative blood transfusion were independently associated with mortality. CONCLUSION: This study highlights a tendency to perform fecal diversion in patients who are acutely ill at presentation. There is a higher morbidity and mortality rate in STM patients. Independent predictors of mortality include CCI, preoperative vasopressor use, steroid use, open abdomen, and intraoperative blood transfusion. Following adjustment by clinical factors, method of colon management was not associated with surgical complications or mortality. LEVEL OF EVIDENCE: Therapeutic study, level IV.

Neutralizing Antibody Induction by HIV-1 Envelope Glycoprotein SOSIP Trimers on Iron Oxide Nanoparticles May Be Impaired by Mannose Binding Lectin.

We covalently attached human immunodeficiency virus type 1 (HIV-1) Env SOSIP trimers to iron oxide nanoparticles (IO-NPs) to create a particulate immunogen for neutralizing antibody (NAb) induction. The attached trimers, ∼20 per particle, retained native-like antigenicity, judged by reactivity with NAbs and non-NAbs. Bivalent (BG505 and B41) trimer IO-NPs were made, as were IO-NPs displaying B41 trimers carrying a PADRE T-cell helper epitope (TCHE). We immunized mice with B41 soluble or IO-NP trimers after PADRE peptide priming. After two immunizations, IO-NP presentation and the TCHE tag independently and substantially increased anti-trimer antibody responses, but titer differences waned after two further doses. Notable and unexpected findings were that autologous NAbs to the N289 glycan hole epitope were consistently induced in mice given soluble but not IO-NP trimers. Various recombinant mannose binding lectins (MBLs) and MBLs in sera of both murine and human origin bound to soluble and IO-NP trimers. MBL binding occluded the autologous NAb epitope on the B41 IO-NP trimers, which may contribute to its poor immunogenicity. The exposure of a subset of broadly active NAb epitopes was also impaired by MBL binding, which could have substantial implications for the utility of trimer-bearing nanoparticles in general and perhaps also for soluble Env proteins.IMPORTANCE Recombinant trimeric SOSIP proteins are vaccine components intended to induce neutralizing antibodies (NAbs) that prevent cells from infection by human immunodeficiency virus type 1 (HIV-1). A way to increase the strength of antibody responses to these proteins is to present them on the surface of nanoparticles (NPs). We chemically attached about 20 SOSIP trimers to NPs made of iron oxide (IO). The resulting IO-NP trimers had appropriate properties when we studied them in the laboratory but, unexpectedly, were less able to induce NAbs than nonattached trimers when used to immunize mice. We found that mannose binding lectins, proteins naturally present in the serum of mice and other animals, bound strongly to the soluble and IO-NP trimers, blocking access to antibody epitopes in a way that may impede the development of NAb responses. These findings should influence how trimer-bearing NPs of various designs are made and used.

Comparative effectiveness analysis of two regional analgesia techniques for the pain management of isolated multiple rib fractures.

Background: Catheter-based regional analgesia has been proposed as an alternative to systemic analgesia for patients with multiple rib fractures (MRF). This study sought to compare the efficacy of regional techniques for decreasing pain and improving clinical outcomes. Study design: This was a multi-institutional, retrospective cohort study of adult (≥18 years) patients admitted to four nonacademic trauma centers over two years (from 07/1/2014 to 06/30/2016). Study inclusion was MRF (≥3 fractures) with no other severe injuries. Two primary regional analgesia techniques were utilized and compared: continuous intercostal nerve blocks (CINB) and epidural (EPI) analgesia. The primary outcome, average pain scores on treatment, was examined using a repeated measures, linear regression mixed model. Secondary outcomes included hospital LOS, ICU LOS, ICU admission and hospital readmission, pulmonary complications, and incentive spirometry volumes during treatment, and were examined with univariate statistics. Results: There were 339 patients with isolated MRF; 85 (25%) required regional analgesia (CINB, n=41; EPI, n=44) and the remaining 75% received systemic analgesia only (IV, n=195; PO, n=59). There were demographic and clinical differences between regional analgesia and systemic analgesia groups; on the contrary, there were no demographic or clinical differences between the CINB and EPI groups. Adjusted pain scores were similar for the EPI and CINB groups (4.0 vs 4.4, p=0.49). Secondary outcomes were worse in the EPI group compared to the CINB group: less improvement in incentive spirometry volume (p=0.004), longer ICU LOS (p=0.03), longer hospital LOS (p<0.001), and more ICU admission (p<0.001). Conclusion: In patients requiring regional analgesia, pain management was equivalent with CINB and EPI, but CINB was associated with significantly better clinical outcomes. CINB might offer an efficient alternative for pain control in patients with MRF.

Optimizing the production and affinity purification of HIV-1 envelope glycoprotein SOSIP trimers from transiently transfected CHO cells.

We describe methods to improve the efficiency with which HIV-1 Envelope glycoprotein SOSIP trimer immunogens can be produced by transient transfection of ExpiCHO-S cells and then affinity purified using the trimer-specific human monoclonal antibody PGT145. The specificity of PGT145 for properly folded trimers allows for the facile, one-step, isolation of these immunogens in research laboratories. PGT145 columns are also valuable as a component of more complex purification processes in current Good Manufacturing Practice programs. However, we found that PGT145 purification was highly variable and markedly inefficient when used to process supernatants from transiently transfected ExpiCHO-S cells expressing the BG505 SOSIP.664 and other trimeric Env proteins. In contrast, no such problems arose when the same Env proteins derived from a stable CHO cell line were processed on the same PGT145 columns, or with transient transfection supernatants from 293F cells. An investigation of the ExpiCHO-S transfection system identified the presence of polyanions, including but perhaps not limited to dextran sulfate, in the Enhancer component of the transfection system. We hypothesized that these polyanions bound to the cationic PGT145 epitope on the trimers and impeded their ability to bind to the PGT145 affinity column. We found that replacing the Enhancer component with alternative culture medium supplements substantially increased the yield of PGT145-purifiable trimers, and we also confirmed that both dextran sulfate and the Enhancer component were indeed inhibitors of PGT145 binding to BG505 SOSIP.664 trimers in immunoassays. The presence of polyanions, including but not limited to nucleic acids, should be considered in other circumstances where PGT145 columns are less efficient than expected at purifying native-like trimers.

Infection after penetrating brain injury-An Eastern Association for the Surgery of Trauma multicenter study oral presentation at the 32nd annual meeting of the Eastern Association for the Surgery of Trauma, January 15-19, 2019, in Austin, Texas.

BACKGROUND: Fatality rates following penetrating traumatic brain injury (pTBI) are extremely high and survivors are often left with significant disability. Infection following pTBI is associated with worse morbidity. The modern rates of central nervous system infections (INF) in civilian survivors are unknown. This study sought to determine the rate of and risk factors for INF following pTBI and to determine the impact of antibiotic prophylaxis. METHODS: Seventeen institutions submitted adult patients with pTBI and survival of more than 72 hours from 2006 to 2016. Patients were stratified by the presence or absence of infection and the use or omission of prophylactic antibiotics. Study was powered at 85% to detect a difference in infection rate of 5%. Primary endpoint was the impact of prophylactic antibiotics on INF. Mantel-Haenszel χ and Wilcoxon's rank-sum tests were used to compare categorical and nonparametric variables. Significance greater than p = 0.2 was included in a logistic regression adjusted for center. RESULTS: Seven hundred sixty-three patients with pTBI were identified over 11 years. 7% (n = 51) of patients developed an INF. Sixty-six percent of INF patients received prophylactic antibiotics. Sixty-two percent of all patients received one dose or greater of prophylactic antibiotics and 50% of patients received extended antibiotics. Degree of dural penetration did not appear to impact the incidence of INF (p = 0.8) nor did trajectory through the oropharynx (p = 0.18). Controlling for other variables, there was no statistically significant difference in INF with the use of prophylactic antibiotics (p = 0.5). Infection was higher in patients with intracerebral pressure monitors (4% vs. 12%; p = <0.001) and in patients with surgical intervention (10% vs. 3%; p < 0.001). CONCLUSION: There is no reduction in INF with prophylactic antibiotics in pTBI. Surgical intervention and invasive intracerebral pressure monitoring appear to be risk factors for INF regardless of prophylactic use. LEVEL OF EVIDENCE: Therapeutic, level IV.

Integrity of Glycosylation Processing of a Glycan-Depleted Trimeric HIV-1 Immunogen Targeting Key B-Cell Lineages.

Broadly neutralizing antibodies (bNAbs) that target the trimeric HIV-1 envelope glycoprotein spike (Env) are tools that can guide the design of recombinant Env proteins intended to engage the predicted human germline precursors of bNAbs (gl-bNAbs). The protein components of gl-bNAb epitopes are often masked by glycans, while mature bNAbs can evolve to accommodate or bypass these shielding glycans. The design of germline-targeting Env immunogens therefore includes the targeted deletion of specific glycan sites. However, the processing of glycans on Env trimers can be influenced by the density with which they are packed together, a highly relevant point given the essential contributions under-processed glycans make to multiple bNAb epitopes. We sought to determine the impact of the removal of 15 potential N-glycan sites (5 per protomer) from the germline-targeting soluble trimer, BG505 SOSIP.v4.1-GT1, using quantitative, site-specific N-glycan mass spectrometry analysis. We find that, compared with SOSIP.664, there was little overall change in the glycan profile but only subtle increases in the extent of processing at sites immediately adjacent to where glycans had been deleted. We conclude that multiple glycans can be deleted from BG505 SOSIP trimers without perturbing the overall integrity of the glycan shield.

Improving the Expression and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers by Targeted Sequence Changes.

Soluble, recombinant native-like envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed for structural studies and as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design is designated SOSIP.664, but many HIV-1 env genes do not yield fully native-like trimers efficiently. One such env gene is CZA97.012 from a neutralization-resistant (tier 2) clade C virus. As appropriately purified, native-like CZA97.012 SOSIP.664 trimers induce autologous neutralizing antibodies (NAbs) efficiently in immunized rabbits, we sought to improve the efficiency with which they can be produced and to better understand the limitations to the original design. By using structure- and antigenicity-guided mutagenesis strategies focused on the V2 and V3 regions and the gp120-gp41 interface, we developed the CZA97 SOSIP.v4.2-M6.IT construct. Fully native-like, stable trimers that display multiple bNAb epitopes could be expressed from this construct in a stable CHO cell line and purified at an acceptable yield using either a PGT145 or a 2G12 bNAb affinity column. We also show that similar mutagenesis strategies can be used to improve the yields and properties of SOSIP.664 trimers of the DU422, 426c, and 92UG037 genotypes.IMPORTANCE Recombinant trimeric proteins based on HIV-1 env genes are being developed for future vaccine trials in humans. A feature of these proteins is their mimicry of the envelope glycoprotein (Env) structure on virus particles that is targeted by neutralizing antibodies, i.e., antibodies that prevent cells from becoming infected. The vaccine concept under exploration is that recombinant trimers may be able to elicit virus-neutralizing antibodies when delivered as immunogens. Because HIV-1 is extremely variable, a practical vaccine may need to incorporate Env trimers derived from multiple different virus sequences. Accordingly, we need to understand how to make recombinant trimers from many different env genes. Here, we show how to produce trimers from a clade C virus, CZA97.012, by using an array of protein engineering techniques to improve a prototypic construct. We also show that the methods may have wider utility for other env genes, thereby further guiding immunogen design.

Síndrome de Fanconi-Bickel / Fanconi-Bickel syndrome

Se presenta el caso de un niño de un año tres meses de edad con síndrome de Fanconi-Bickel, caracterizado por las manifestaciones del síndrome de Fanconi (glucosuria, aminoaciduria y fosfaturia) y acúmulo de glucógeno en el hígado semejante a lo que se observa en la glucogenosis tipo Ia. Debido a la presencia de glucogenosis hepática el paciente también presentó hipoglucemia, cetonuria, hipercolesterolemia e hipertrigliceridemia. La glucogenosis observada en los pacientes con síndrome de Fanconi-Bickel, no depende de defecto de la actividad de la glucosa-6-fosfatasa, sino al parecer, de un defecto del transportador que moviliza la glucosa y la galactosa en el hígado y en la membrana basolateral del túbulo proximal del nefrón