Insights into next generation sequencing guided antibody selection strategies.

Therapeutic antibody discovery often relies on in-vitro display methods to identify lead candidates. Assessing selected output diversity traditionally involves random colony picking and Sanger sequencing, which has limitations. Next-generation sequencing (NGS) offers a cost-effective solution with increased read depth, allowing a comprehensive understanding of diversity.

Brilacidin, a Non-Peptide Defensin-Mimetic Molecule, Inhibits SARS-CoV-2 Infection by Blocking Viral Entry.

Brilacidin (PMX-30063), a non-peptide defensin-mimetic small molecule, inhibits SARS-CoV-2 viral infection but the anti-viral mechanism is not defined. Here we determined its effect on the specific step of the viral life cycle. Brilacidin blocked SARS-CoV-2 infection but had no effect after viral entry.

A pandemic-enabled comparison of discovery platforms demonstrates a naïve antibody library can match the best immune-sourced antibodies.

As a result of the SARS-CoV-2 pandemic numerous scientific groups have generated antibodies against a single target: the CoV-2 spike antigen. This has provided an unprecedented opportunity to compare the efficacy of different methods and the specificities and qualities of the antibodies generated by those methods. Generally, the most potent neutralizing antibodies have been generated from convalescent patients and immunized animals, with non-immune phage libraries usually yielding significantly less potent antibodies.

Robust IgM responses following intravenous vaccination with Bacille Calmette-Guérin associate with prevention of Mycobacterium tuberculosis infection in macaques.

Development of an effective tuberculosis (TB) vaccine has suffered from an incomplete understanding of the correlates of protection against Mycobacterium tuberculosis (Mtb). Intravenous (i.v.

Determinants and Dynamics of SARS-CoV-2 Infection in a Diverse Population: 6-Month Evaluation of a Prospective Cohort Study.

Background: We studied risk factors, antibodies, and symptoms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in a diverse, ambulatory population.

Methods: A prospective cohort (n = 831) previously undiagnosed with SARS-CoV-2 infection underwent serial testing (SARS-CoV-2 polymerase chain reaction, immunoglobulin G [IgG]) for 6 months.

Results: Ninety-three participants (11.

Human Immunodeficiency Viruses Pseudotyped with SARS-CoV-2 Spike Proteins Infect a Broad Spectrum of Human Cell Lines through Multiple Entry Mechanisms.

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19), enters cells through attachment to the human angiotensin converting enzyme 2 (hACE2) via the receptor-binding domain (RBD) in the surface/spike (S) protein. Several pseudotyped viruses expressing SARS-CoV-2 S proteins are available, but many of these can only infect hACE2-overexpressing cell lines. Here, we report the use of a simple, two-plasmid, pseudotyped virus system comprising a SARS-CoV-2 spike-expressing plasmid and an HIV vector with or without to investigate the SARS-CoV-2 entry event in various cell lines.

Vaccination boosts protective responses and counters SARS-CoV-2-induced pathogenic memory B cells.

Much is to be learned about the interface between immune responses to SARS-CoV-2 infection and vaccination. We monitored immune responses specific to SARS-CoV-2 Spike Receptor-Binding-Domain (RBD) in convalescent individuals for eight months after infection diagnosis and following vaccination. Over time, neutralizing antibody responses, which are predominantly RBD specific, generally decreased, while RBD-specific memory B cells persisted.

Lipoarabinomannan antigenic epitope differences in tuberculosis disease subtypes.

An accurate urine test for diverse populations with active tuberculosis could be transformative for preventing TB deaths. Urinary liporabinomannan (LAM) testing has been previously restricted to HIV co-infected TB patients. In this study we evaluate urinary LAM in HIV negative, pediatric and adult, pulmonary and extrapulmonary tuberculosis patients.

A Novel Sensitive Immunoassay Targeting the 5-Methylthio-d-Xylofuranose-Lipoarabinomannan Epitope Meets the WHO's Performance Target for Tuberculosis Diagnosis.

The only currently commercialized point-of-care assay for tuberculosis (TB) that measures lipoarabinomannan (LAM) in urine (Alere LF-LAM) has insufficient sensitivity. We evaluated the potential of 100 novel monoclonal antibody pairs targeting a variety of LAM epitopes on a sensitive electrochemiluminescence platform to improve the diagnostic accuracy. In the screening, many antibody pairs showed high reactivity to purified LAM but performed poorly at detecting urinary LAM in clinical samples, suggesting differences in antigen structure and immunoreactivity of the different LAM sources.

Detection of lipoarabinomannan in urine and serum of HIV-positive and HIV-negative TB suspects using an improved capture-enzyme linked immuno absorbent assay and gas chromatography/mass spectrometry.

TB diagnosis and treatment monitoring in resource limited regions rely heavily on serial sputum smear microscopy and bacterial culture. These microbiological methods are time-consuming, expensive and lack adequate sensitivity. The WHO states that improved TB diagnosis and treatment is imperative to achieve an end to the TB epidemic by 2030.

Characterization of the Antigenic Heterogeneity of Lipoarabinomannan, the Major Surface Glycolipid of , and Complexity of Antibody Specificities toward This Antigen.

Lipoarabinomannan (LAM), the major antigenic glycolipid of , is an important immunodiagnostic target for detecting tuberculosis (TB) infection in HIV-1-coinfected patients, and is believed to mediate a number of functions that promote infection and disease development. To probe the human humoral response against LAM during TB infection, several novel LAM-specific human mAbs were molecularly cloned from memory B cells isolated from infected patients and grown in vitro. The fine epitope specificities of these Abs, along with those of a panel of previously described murine and phage-derived LAM-specific mAbs, were mapped using binding assays against LAM Ags from several mycobacterial species and a panel of synthetic glycans and glycoconjugates that represented diverse carbohydrate structures present in LAM.

Identification of Novel Structural Determinants in MW965 Env That Regulate the Neutralization Phenotype and Conformational Masking Potential of Primary HIV-1 Isolates.

The subtype C HIV-1 isolate MW965.26 is a highly neutralization-sensitive tier 1a primary isolate that is widely used in vaccine studies, but the basis for the sensitive neutralization phenotype of this isolate is not known. Substituting the MW965.

Specific sequences commonly found in the V3 domain of HIV-1 subtype C isolates affect the overall conformation of native Env and induce a neutralization-resistant phenotype independent of V1/V2 masking.

Primary HIV-1 isolates are relatively resistant to neutralization by antibodies commonly induced after infection or vaccination. This is generally attributed to masking of sensitive epitopes by the V1/V2 domain and/or glycans situated at various positions in Env. Here we identified a novel masking effect mediated by subtype C-specific V3 sequences that contributes to the V1/V2-independent and glycan-independent neutralization resistance of chimeric and primary Envs to antibodies directed against multiple neutralization domains.

Characterization of structural features and diversity of variable-region determinants of related quaternary epitopes recognized by human and rhesus macaque monoclonal antibodies possessing unusually potent neutralizing activities.

A series of potently neutralizing monoclonal antibodies (MAbs) that target quaternary epitopes on the native Env trimer have recently been described. A common feature shared by these antibodies is the critical involvement of sites in both the V2 and V3 variable domains in antibody recognition. In this study the gp120 variable-region determinants were mapped for eight rhesus macaque monoclonal antibodies (RhMAbs) possessing potently neutralizing activity specific for a quaternary target in SF162 Env and compared to those originally identified for human MAb 2909.

Potent and broad neutralization of HIV-1 subtype C by plasma antibodies targeting a quaternary epitope including residues in the V2 loop.

The targets of broadly cross-neutralizing (BCN) antibodies are of great interest in the HIV vaccine field. We have identified a subtype C HIV-1-superinfected individual, CAP256, with high-level BCN activity, and characterized the antibody specificity mediating breadth. CAP256 developed potent BCN activity peaking at 3 years postinfection, neutralizing 32 (76%) of 42 heterologous viruses, with titers of antibodies against some viruses exceeding 1:10,000.

Subtype-specific conservation of isoleucine 309 in the envelope V3 domain is linked to immune evasion in subtype C HIV-1 infection.

The V3 region of the HIV-1 envelope (Env) glycoprotein gp120 is a key functional domain yet it exhibits distinct mutational patterns across subtypes. Here an invariant residue (Ile 309) was replaced with Leu in 7 subtype C patient-derived Envs from recent infection and 4 related neutralizing antibody escape variants that emerged later. For these 11 Envs, I309L did not alter replication in primary CD4 T cells; however, replication in monocyte-derived macrophages was enhanced.

The C108g epitope in the V2 domain of gp120 functions as a potent neutralization target when introduced into envelope proteins derived from human immunodeficiency virus type 1 primary isolates.

Monoclonal antibodies (MAbs) directed against epitopes in the V2 domain of human immunodeficiency virus type 1 gp120 often possess neutralizing activity, but these generally are highly type specific, neutralize only laboratory isolates, or have low potency. The most potent of these is C108g, directed against a type-specific epitope in HXB2 and BaL gp120s, which is glycan dependent and, in contrast to previous reports, dependent on intact disulfide bonds. This epitope was introduced into two primary Envs, derived from a neutralization-sensitive (SF162) and a neutralization-resistant (JR-FL) isolate, by substitution of two residues and, for SF162, addition of an N-linked glycosylation site.