Altered Env conformational dynamics as a mechanism of resistance to peptide-triazole HIV-1 inactivators.

BACKGROUND: We previously developed drug-like peptide triazoles (PTs) that target HIV-1 Envelope (Env) gp120, potently inhibit viral entry, and irreversibly inactivate virions. Here, we investigated potential mechanisms of viral escape from this promising class of HIV-1 entry inhibitors. RESULTS: HIV-1 resistance to cyclic (AAR029b) and linear (KR13) PTs was obtained by dose escalation in viral passaging experiments. High-level resistance for both inhibitors developed slowly (relative to escape from gp41-targeted C-peptide inhibitor C37) by acquiring mutations in gp120 both within (Val255) and distant to (Ser143) the putative PT binding site. The similarity in the resistance profiles for AAR029b and KR13 suggests that the shared IXW pharmacophore provided the primary pressure for HIV-1 escape. In single-round infectivity studies employing recombinant virus, V255I/S143N double escape mutants reduced PT antiviral potency by 150- to 3900-fold. Curiously, the combined mutations had a much smaller impact on PT binding affinity for monomeric gp120 (four to ninefold). This binding disruption was entirely due to the V255I mutation, which generated few steric clashes with PT in molecular docking. However, this minor effect on PT affinity belied large, offsetting changes to association enthalpy and entropy. The escape mutations had negligible effect on CD4 binding and utilization during entry, but significantly altered both binding thermodynamics and inhibitory potency of the conformationally-specific, anti-CD4i antibody 17b. Moreover, the escape mutations substantially decreased gp120 shedding induced by either soluble CD4 or AAR029b. CONCLUSIONS: Together, the data suggest that the escape mutations significantly modified the energetic landscape of Env's prefusogenic state, altering conformational dynamics to hinder PT-induced irreversible inactivation of Env. This work therein reveals a unique mode of virus escape for HIV-1, namely, resistance by altering the intrinsic conformational dynamics of the Env trimer.

Adenosine deaminase augments SARS-CoV-2 specific cellular and humoral responses in aged mouse models of immunization and challenge.

Despite numerous clinically available vaccines and therapeutics, aged patients remain at increased risk for COVID-19 morbidity. Furthermore, various patient populations, including the aged can have suboptimal responses to SARS-CoV-2 vaccine antigens. Here, we characterized vaccine-induced responses to SARS-CoV-2 synthetic DNA vaccine antigens in aged mice. Aged mice exhibited altered cellular responses, including decreased IFNγ secretion and increased TNFα and IL-4 secretion suggestive of TH2-skewed responses. Aged mice exhibited decreased total binding and neutralizing antibodies in their serum but significantly increased TH2-type antigen-specific IgG1 antibody compared to their young counterparts. Strategies to enhance vaccine-induced immune responses are important, especially in aged patient populations. We observed that co-immunization with plasmid-encoded adenosine deaminase (pADA)enhanced immune responses in young animals. Ageing is associated with decreases in ADA function and expression. Here, we report that co-immunization with pADA enhanced IFNγ secretion while decreasing TNFα and IL-4 secretion. pADA expanded the breadth and affinity SARS-CoV-2 spike-specific antibodies while supporting TH1-type humoral responses in aged mice. scRNAseq analysis of aged lymph nodes revealed that pADA co-immunization supported a TH1 gene profile and decreased FoxP3 gene expression. Upon challenge, pADA co-immunization decreased viral loads in aged mice. These data support the use of mice as a model for age-associated decreased vaccine immunogenicity and infection-mediated morbidity and mortality in the context of SARS-CoV-2 vaccines and provide support for the use of adenosine deaminase as a molecular adjuvant in immune-challenged populations.

Characterization of neutralizing affinity-matured human respiratory syncytial virus F binding antibodies in the sub-picomolar affinity range.

In the human adaptation and optimization of a mouse anti-human respiratory syncytial virus neutralizing antibody, affinity assessment was crucial to distinguish among potential candidates and to evaluate whether this correlated with function in vitro and in vivo. This affinity assessment was complicated by the trimeric nature of the antigen target, respiratory syncytial virus F (RSV-F) glycoprotein. In the initial affinity screen, surface plasmon resonance was used to determine the intrinsic binding affinities of anti-RSV-F Fab and immunoglobulin G (IgG) to the extracellular domain of RSV-F. This assessment required minimal biotinylation of the RSV-F protein and design of a capture strategy to minimize avidity effects. Approximately 30 Fabs were selected from three optimization phage display libraries on the basis of an initial ELISA screen. Surface plasmon resonance analysis demonstrated the success of optimization with some candidates from the screened libraries having low picomolar dissociation constants, more than 700-fold tighter than the parental monoclonal antibody (B21M). The affinities of these antibodies were further evaluated by a kinetic exclusion assay, a solution binding technology. One IgG (monoclonal antibody 029) displayed a low picomolar K(D) comparable with that of motavizumab, an RSV antibody in clinical study. Kinetic exclusion assay showed that two other of the matured IgGs (011 and 019) had sub-picomolar dissociation constants that could not be resolved further. We discuss the relevance of these interaction analysis results in the light of recently published data on the mechanism of F-driven viral fusion during paramyxoviral infection and 101F epitope conservation revealed from the recent crystal structure of RSV-F in the post-fusion state.

The Case for S2: The Potential Benefits of the S2 Subunit of the SARS-CoV-2 Spike Protein as an Immunogen in Fighting the COVID-19 Pandemic.

As COVID-19 cases continue to rise, it is imperative to learn more about antibodies and T-cells produced against the causative virus, SARS-CoV-2, in order to guide the rapid development of therapies and vaccines. While much of the current antibody and vaccine research focuses on the receptor-binding domain of S1, a less-recognized opportunity is to harness the potential benefits of the more conserved S2 subunit. Similarities between the spike proteins of both SARS-CoV-2 and HIV-1 warrant exploring S2. Possible benefits of employing S2 in therapies and vaccines include the structural conservation of S2, extant cross-reactive neutralizing antibodies in populations (due to prior exposure to common cold coronaviruses), the steric neutralization potential of antibodies against S2, and the stronger memory B-cell and T-cell responses. More research is necessary on the effect of glycans on the accessibility and stability of S2, SARS-CoV-2 mutants that may affect infectivity, the neutralization potential of antibodies produced by memory B-cells, cross-reactive T-cell responses, antibody-dependent enhancement, and antigen competition. This perspective aims to highlight the evidence for the potential advantages of using S2 as a target of therapy or vaccine design.

High blood pressure, overweight and obesity among rural scholars from the Vela Project: a population-based study from South America.

BACKGROUND: Many studies have shown that high blood pressure and overweight begins in childhood. Consequently, it is useful to know blood pressure and body mass index (BMI) values from an early age. There are few data about blood pressure control in children and adolescents from rural populations in South America. AIM: The objective of this study was to determine the prevalence of high blood pressure and its association with sedentary habits and overweight/obesity in scholars from a rural population in Argentina. METHODS: The study population for this cross-sectional study was composed of rural children and adolescent scholars from Maria Ignacia Vela. Pre-hypertension and hypertension were defined on the basis of percentiles from the average of three blood pressure measurements taken on a single occasion. In patients with three blood pressure measurements above the 90th percentile, ambulatory blood pressure monitoring was performed to confirm hypertension or pre-hypertension. BMI was categorized by using the 2000 Centers for Disease Control and Prevention growth charts. RESULTS: We studied 334 scholars (aged 5-18 years). Mean age was 11.4 years. In 70% of the subjects, blood pressure had never been measured. The prevalence of high blood pressure was 4.4%. Students with sedentary habits were 3.67-fold more likely to develop high blood pressure than their physically active counterparts (odds ratio [OR] 3.67; 95% CI 1.08, 12.46; p = 0.037). Obese students were more likely to develop hypertension than the students with normal weight (OR = 5.17; 95% CI 1.52, 17.60; p = 0.02). Male students had a 3.4-fold higher risk of developing high blood pressure than females. CONCLUSIONS: In our rural population, the evaluation of blood pressure in children and adolescents is not a routine measure. Our data indicate a low prevalence of high blood pressure. These data could argue differences between rural and urban scholars. Our data demonstrate a close relationship between increased overweight, obesity and sedentary lifestyle with the development of high blood pressure. We emphasize the importance of blood pressure controls and the need to implement programmes to modify sedentary lifestyle in rural populations.

Kinetic screening of antibodies from crude hybridoma samples using Biacore.

Experimental and data analysis protocols were developed to screen antibodies from hybridoma culture supernatants using Biacore surface plasmon resonance biosensor platforms. The screening methods involved capturing antibodies from crude supernatants using Fc-specific antibody surfaces and monitoring antigen binding at a single concentration. After normalizing the antigen responses for the amount of antibody present, a simple interaction model was fit to all of the binding responses simultaneously. As a result, the kinetic rate constants (k(a) and k(d)) and affinity (K(D)) could be determined for each antibody interaction under identical conditions. Higher-resolution studies involving multiple concentrations of antigen were performed to validate the reliability of single-concentration measurements. The screening protocols can be used to characterize antigen binding kinetics to approximately 200 antibody supernatants per day using automated Biacore 2000 and 3000 instruments.

Biosensor analysis of dynamics of interleukin 5 receptor subunit beta(c) interaction with IL5:IL5R(alpha) complexes.

To gain insight into IL5 receptor subunit recruitment mechanism, and in particular the experimentally elusive pathway for assembly of signaling subunit beta(c), we constructed a soluble beta(c) ectodomain (s(beta)(c)) and developed an optical biosensor assay to measure its binding kinetics. Functionally active s(beta)(c) was anchored via a C-terminal His tag to immobilized anti-His monoclonal antibodies on the sensor surface. Using this surface, we quantitated for the first time direct binding of s(beta)(c) to IL5R(alpha) complexed to either wild-type or single-chain IL5. Binding was much weaker if at all with either R(alpha) or IL5 alone. Kinetic evaluation revealed a moderate affinity (0.2-1 microM) and relatively fast off rate for the s(beta)(c) interaction with IL5:R(alpha) complexes. The data support a model in which beta(c) recruitment occurs with preformed IL5:R(alpha) complex. Dissociation kinetics analysis suggests that the IL5-alpha-beta(c) complex is relatively short-lived. Overall, this study solidifies a model of sequential recruitment of receptor subunits by IL5, provides a novel biosensor binding assay of beta(c) recruitment dynamics, and sets the stage for more advanced characterization of the roles of structural elements within R(alpha), beta(c), and cytokines of the IL5/IL3/GM-CSF family in receptor recruitment and activation.

Beta-turn Phe in HIV-1 Env binding site of CD4 and CD4 mimetic miniprotein enhances Env binding affinity but is not required for activation of co-receptor/17b site.

HIV-1 enters a host cell after an initial interaction between viral envelope glycoprotein gp120 and cell surface receptor CD4, followed by a second interaction between gp120 and a cell surface chemokine receptor. CD4 residue Phe43 makes a significant contribution to the high-affinity interaction between CD4 and env. We and others have used scorpion toxin scaffolds to display and examine CD4 epitopes used for gp120 recognition. These peptides, which have a beta-turn Phe that acts as a Phe43 surrogate, compete with CD4 for gp120 binding and enhance the binding of gp120 to 17b, an antibody that binds near the co-receptor-binding site. In the current study, a scyllatoxin-scaffolded peptide, identified via phage epitope randomization and lacking a beta-turn Phe (indeed, containing no aromatic residues), was shown to behave in a distinctly CD4-like manner. This peptide, denoted [20EGLV23]ST, not only competed with CD4 for gp120 binding, but also enhanced the binding of gp120 to 17b. Quantitatively, an [20EGLV23]ST-gp120 complex exhibited the same 17b binding on-rate as a complex of gp120 with [20AGSF23]ST, a scyllatoxin-based CD4 mimetic peptide containing a beta-turn Phe. In view of this result, we examined the role of Phe43 in CD4 itself by comparing F43V D1D2 sCD4 versus D1D2 sCD4. Like the peptides, a close similarity was observed for both Phe43 and Phe43-less D1D2 sCD4s in enhancing gp120 binding to 17b. Further, when examined for their ability to enhance binding of gp120 to CCR5+ cells, [20EGLV23]ST and [20AGSF23]ST were found to have the same efficacy, after correcting for the difference in their gp120 affinities. These results show that, although Phe43 is important in maintaining high affinity in gp120 ligands, the aromatic residue is not necessary for triggering the conformational isomerization in gp120 that results in formation or exposure of the binding sites for the 17b antibody and the CCR5 receptor.

Resistencia de Crotalus durissus terificus y Bothrops neuwiedii a la neurotoxidad de cantidades masivas de veneno crotalico. / Resistance of Crotalus durissus terrificus and Bothrops neuwiedii to neurotoxicity of massive quantities of Crotalid vevenom

Especimenes de Crotalus durissus terrificus y de Bothrops neuwiedii toleran cantidades de veneno crotalico equivalentes a 5 x 10 elevado a menos 4 de su peso.La tolerancia observada puede explicarse por la presencia en el suero de ambas especies de componente (s) capaces de inactivar crotoxina, la neurotoxina mayor del veneno crotalico. La potencia antitoxica del suero antitoxica del suero de C. d. terrificus frente al veneno crotalico es similar a la del suero antiofidico monovalente obtenido de caballo hiperinmune, mientras que la potencia antitoxica del suero de B. neuwiedii es de alredor del 20% del de este ultimo. Los sueros de ambas especies en concentraciones adequadas son capaces de de proteger al raton frente a 4 LD 50 de veneno crotalico. La ausencia de lineas de precipitacion en ensayo de doble difusion de suero frente a veneno sugiere que los factores antitoxicos de ambas especies no son inmunoglobulinas. Es probable que la crotoxina sea neutralizada por la formacion de complexos inactivos con componentes especificos del suero. La resistencia al veveno no es reciproca, ya que especimenes de C.d. terrificus mueren luego de la inyeccion de cantidades de veneno de B.neuwiedii que son perfectamente toleradas por la especie dadora

Resistencia de Crotalus durissus terificus y Bothrops neuwiedii a la neurotoxidad de cantidades masivas de veneno crotalico. / Resistance of Crotalus durissus terrificus and Bothrops neuwiedii to neurotoxicity of massive quantities of Crotalid vevenom

Especimenes de Crotalus durissus terrificus y de Bothrops neuwiedii toleran cantidades de veneno crotalico equivalentes a 5 x 10 elevado a menos 4 de su peso.La tolerancia observada puede explicarse por la presencia en el suero de ambas especies de componente (s) capaces de inactivar crotoxina, la neurotoxina mayor del veneno crotalico. La potencia antitoxica del suero antitoxica del suero de C. d. terrificus frente al veneno crotalico es similar a la del suero antiofidico monovalente obtenido de caballo hiperinmune, mientras que la potencia antitoxica del suero de B. neuwiedii es de alredor del 20% del de este ultimo. Los sueros de ambas especies en concentraciones adequadas son capaces de de proteger al raton frente a 4 LD 50 de veneno crotalico. La ausencia de lineas de precipitacion en ensayo de doble difusion de suero frente a veneno sugiere que los factores antitoxicos de ambas especies no son inmunoglobulinas. Es probable que la crotoxina sea neutralizada por la formacion de complexos inactivos con componentes especificos del suero. La resistencia al veveno no es reciproca, ya que especimenes de C.d. terrificus mueren luego de la inyeccion de cantidades de veneno de B.neuwiedii que son perfectamente toleradas por la especie dadora