ВЛИЯНИЕ НАНОЧАСТИЦ ЗОЛОТА НА АГРЕГАЦИЮ МИТОХОНДРИАЛЬНОЙ АСПАРТАТ-АМИНОТРАНСФЕРАЗЫ

We have studied the dependence of aggregation of mitochondrial aspartate aminotransferase (mAspAT) from the concentration of gold nanoparticles (AuNP). It has been shown that AuNPs decreased the aggregation of mAspAT in the temperature range from 55 to 73 °C. The maximal anti-aggregational effect of AuNP reached 56 % and was observed at 60 °C. Increase of AuNP concentration led to a decrease of the constant rate of enzyme aggregation. We suggest here that interaction between Au-NPs and mAspAT increases conformational stability of the enzyme molecule. It also reduces the probability of polypeptide chain unfolding, which causes exposure of hydrophobic patches on the protein surface resulting in intra molecular adhesion followed by the protein aggregation.Исследована зависимость агрегации митохондриальной аспартат-аминотрансферазы (мАспАТ) от концентрации наночастиц золота (НЧЗ). Установлено, что в температурном диапазоне от 55 до 73 °С НЧЗ снижают агрегацию мАспАТ. Наиболее выраженный антиагрегационный эффект НЧЗ достигает 56 % и проявляется при 60 °С. Увеличение концентрации НЧЗ приводит к снижению константы скорости агрегации фермента. Высказывается предположение, что формирование связей между НЧЗ и мАспАТ будет приводить к повышению конформационной жесткости молекулы фермента, снижать вероятность анфолдинга и экспонирования на поверхности молекулы гидрофобных фрагментов, обеспечивающих межмолекулярное «слипание» и последующую агрегацию белка

Serological fingerprints link antiviral activity of therapeutic antibodies to affinity and concentration

The effectiveness of therapeutic monoclonal antibodies (mAbs) against variants of the SARS-CoV-2 virus is highly variable. As target recognition of mAbs relies on tight binding affinity, we assessed the affinities of five therapeutic mAbs to the receptor binding domain (RBD) of wild type (A), Delta (B.1.617.2), and Omicron BA.1 SARS-CoV-2 (B.1.1.529.1) spike using microfluidic diffusional sizing (MDS). Four therapeutic mAbs showed strongly reduced affinity to Omicron BA.1 RBD, whereas one (sotrovimab) was less impacted. These affinity reductions correlate with reduced antiviral activities suggesting that affinity could serve as a rapid indicator for activity before time-consuming virus neutralization assays are performed. We also compared the same mAbs to serological fingerprints (affinity and concentration) obtained by MDS of antibodies in sera of 65 convalescent individuals. The affinities of the therapeutic mAbs to wild type and Delta RBD were similar to the serum antibody response, indicating high antiviral activities. For Omicron BA.1 RBD, only sotrovimab retained affinities within the range of the serum antibody response, in agreement with high antiviral activity. These results suggest that serological fingerprints provide a route to evaluating affinity and antiviral activity of mAb drugs and could guide the development of new therapeutics

Defense-in-depth by mucosally administered anti-HIV dimeric IgA2 and systemic IgG1 mAbs: complete protection of rhesus monkeys from mucosal SHIV challenge.

Although IgA is the most abundantly produced immunoglobulin in humans, its role in preventing HIV-1 acquisition, which occurs mostly via mucosal routes, remains unclear. In our passive mucosal immunizations of rhesus macaques (RMs), the anti-HIV-1 neutralizing monoclonal antibody (nmAb) HGN194, given either as dimeric IgA1 (dIgA1) or dIgA2 intrarectally (i.r.), protected 83% or 17% of the RMs against i.r. simian-human immunodeficiency virus (SHIV) challenge, respectively. Data from the RV144 trial implied that vaccine-induced plasma IgA counteracted the protective effector mechanisms of IgG1 with the same epitope specificity. We thus hypothesized that mucosal dIgA2 might diminish the protection provided by IgG1 mAbs targeting the same epitope. To test our hypothesis, we administered HGN194 IgG1 intravenously (i.v.) either alone or combined with i.r. HGN194 dIgA2. We enrolled SHIV-exposed, persistently aviremic RMs protected by previously administered nmAbs; RM anti-human IgG responses were undetectable. However, low-level SIV Gag-specific proliferative T-cell responses were found. These animals resemble HIV-exposed, uninfected humans, in which local and systemic cellular immune responses have been observed. HGN194 IgG1 and dIgA2 used alone and the combination of the two neutralized the challenge virus equally well in vitro. All RMs given only i.v. HGN194 IgG1 became infected. In contrast, all RMs given HGN194 IgG1+dIgA2 were completely protected against high-dose i.r. SHIV-1157ipEL-p challenge. These data imply that combining suboptimal defenses at the mucosal and systemic levels can completely prevent virus acquisition. Consequently, active vaccination should focus on defense-in-depth, a strategy that seeks to build up defensive fall-back positions well behind the fortified frontline.We thank Dr. J. Mascola for providing mAb VRC01, Dr. S.-L. Hu for providing SHIV-1157ip Env proteins, and Dr. W. Marasco for providing mAb Fm-6. We thank Dr. K. Rogers and K. Kinsley for TRIM5α genotype analysis, Dr. S. Lee for assistance in statistical analysis, V. Shanmuganathan for technical assistance, and Juan Esquivel for assistance with the preparation of the manuscript. This was work supported by the Bill and Melinda Gates Foundation Collaboration for AIDS Vaccine Discovery (CAVD) UCL-VDC Grant 38637 (R.A.W.). This project was also funded in part by NIH grants P01 AI048240, R01 AI100703 and R37 AI034266 to RMR. Base grant P51 OD011132 provided support to the Yerkes National Primate Research Center. The Southwest National Primate Research Center is supported by an NIH primate center base grant (previously NCRR grant P51 RR013986; currently Office of Research Infrastructure Programs/OD P51 OD011133).This is the accepted manuscript of a paper published in Vaccine (Sholukh AM, et al., Vaccine, 2015, 33, 2086-2095, doi:10.1016/j.vaccine.2015.02.020). The final version is available at http://dx.doi.org/10.1016/j.vaccine.2015.02.02

Passive immunization of macaques with polyclonal anti-SHIV IgG against a heterologous tier 2 SHIV: outcome depends on IgG dose

Background: A key goal for HIV-1 envelope immunogen design is the induction of cross-reactive neutralizing antibodies (nAbs). As AIDS vaccine recipients will not be exposed to strains exactly matching any immunogens due to multiple HIV-1 quasispecies circulating in the human population worldwide, heterologous SHIV challenges are essential for realistic vaccine efficacy testing in primates. We assessed whether polyclonal IgG, isolated from rhesus monkeys (RMs) with high-titer nAbs (termed SHIVIG), could protect RMs against the R5-tropic tier-2 SHIV-2873Nip, which was heterologous to the viruses or HIV-1 envelopes that had elicited SHIVIG. Results: SHIVIG demonstrated binding to HIV Gag, Tat, and Env of different clades and competed with the broadly neutralizing antibodies b12, VRC01, 4E10, and 17b. SHIVIG neutralized tier 1 and tier 2 viruses, including SHIV-2873Nip. NK-cell depletion decreased the neutralizing activity of SHIVIG 20-fold in PBMC assays. Although SHIVIG neutralized SHIV-2873Nip in vitro, this polyclonal IgG preparation failed to prevent acquisition after repeated intrarectal low-dose virus challenges, but at a dose of 400 mg/kg, it significantly lowered peak viremia (P = 0.001). Unexpectedly, single-genome analysis revealed a higher number of transmitted variants at the low dose of 25 mg/kg, implying increased acquisition at low SHIVIG levels. In vitro, SHIVIG demonstrated complement-mediated Ab-dependent enhancement of infection (C’-ADE) at concentrations similar to those observed in plasmas of RMs treated with 25 mg/kg of SHIVIG. Conclusion: Our primate model data suggest a dual role for polyclonal anti-HIV-1 Abs depending on plasma levels upon virus encounter

An Anti-HIV-1 V3 Loop Antibody Fully Protects Cross-Clade and Elicits T-Cell Immunity in Macaques Mucosally Challenged with an R5 Clade C SHIV

Neutralizing antibodies have been shown to protect macaques against SHIV challenge. However, genetically diverse HIV-1 clades have evolved, and a key question left unanswered is whether neutralizing antibodies can confer cross-clade protection in vivo. The novel human monoclonal antibody HGN194 was isolated from an individual infected with an HIV-1 clade AG recombinant circulating recombinant form (CRF). HGN194 targets an epitope in the third hypervariable loop (V3) of HIV-1 gp120 and neutralizes a range of relatively neutralization-sensitive and resistant viruses. We evaluated the potential of HGN194 to protect infant rhesus monkeys against a SHIV encoding a primary CCR5-tropic HIV-1 clade C envelope. After high-dose mucosal challenge, all untreated controls became highly viremic while all HGN194-treated animals (50 mg/kg) were completely protected. When HGN194 was given at 1 mg/kg, one out of two monkeys remained aviremic, whereas the other had delayed, lower peak viremia. Interestingly, all protected monkeys given high-dose HGN194 developed Gag-specific proliferative responses of both CD4+ and CD8+ T cells. To test whether generation of the latter involved cryptic infection, we ablated CD8+ cells after HGN194 clearance. No viremia was detected in any protected monkeys, thus ruling out virus reservoirs. Thus, induction of CD8 T-cell immunity may have resulted from transient “Hit and Run” infection or cross priming via Ag-Ab-mediated cross-presentation. Together, our data identified the HGN194 epitope as protective and provide proof-of-concept that this anti-V3 loop mAb can prevent infection with sterilizing immunity after challenge with virus of a different clade, implying that V3 is a potential vaccine target

Влияние элементного состава и температуры осаждения покрытий Ti-AL-C-N на их морфологию и жизнеспособность клеток на таких покрытиях

Nanostructural Ti-Al-C-N coatings were produced by reactive magnetron sputtering at substrate temperatures of 220, 340 and 440 °C using mosaic targets with different Al/Ti ratios. Using atomic-force and scanning electron microscopy, it was found that the variation of the elemental composition leads to a change in the morphology of Ti-Al-C-N coatings: at an Al/Ti ratio of 0.39, the films have a mixed columnar-granular structure with no visible defects and a low roughness (3.30–5.86 nm); at an Al/Ti ratio of ~ 0.96, the films show a porous columnar structure with a higher roughness (8.83–11.07 nm) and for an Al/Ti ratio of ~ 1.71, the films have a fine-grained structure and the smallest roughness values (0.48–1.74 nm). Substrate heating from 220 to 440 °C did not significantly affect the elemental composition of Ti-Al-C-N films, but it affected the deposition rate, surface roughness, and the microstructure of the coatings. MTT-test results showed no relationship between the fibroblasts viability, the coating roughness and the coating elemental composition. However, the cells viability and their ability to proliferate on the Ti-Al-C-N coatings surface were preserved.Наноструктурные покрытия Ti-Al-C-N формировались методом реактивного магнетронного осаждения из мозаичных мишеней с различным соотношением Al/Ti при температурах подложки 220, 340 и 440 °C. Методами атомно-силовой и растровой электронной микроскопии обнаружено, что варьирование элементного состава приводит к изменению морфологии покрытий Ti-Al-C-N: при соотношении Al/Ti ~ 0,39 пленки имеют столбчато-зернистую структуру без видимых дефектов и низкую шероховатость Sq  (3,30–5,86 нм); при Al/Ti ~ 0,96 пленки показали столбчатую пористую структуру и более высокую шероховатость Sq (8,83–11,07 нм); при Al/Ti ~ 1,71 имели мелкозернистую структуру и наименьшие значения шероховатости Sq  (0,48–1,74 нм). Нагрев подложки от 220 до 440 °C не оказывал значительного влияния на элементный состав Ti-Al-C-N пленок, однако воздействовал на скорость осаждения, шероховатость поверхности и микроструктуру покрытий. По результатам МТТ-теста прямой зависимости между жизнеспособностью фибробластов, шероховатостью покрытий и их элементным составом не обнаружено, однако жизнеспособность клеток и их способность к пролиферации при контакте с поверхностью покрытий Ti-Al-C-N сохранялась

Isolation of Monoclonal Antibodies with Predetermined Conformational Epitope Specificity

Existing technologies allow isolating antigen-specific monoclonal antibodies (mAbs) from B cells. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes) that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs) that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs). To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. We established proof-of-concept of our strategy by isolating mAbs targeting the conformational V3 loop crown of HIV Env; the new mAbs cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents

An anti-HIV-1 V3 loop antibody fully protects cross-clade and elicits T-cell immunity in macaques mucosally challenged with an R5 clade C SHIV

Neutralizing antibodies have been shown to protect macaques against SHIV challenge. However, genetically diverse HIV-1 clades have evolved, and a key question left unanswered is whether neutralizing antibodies can confer cross-clade protection in vivo. The novel human monoclonal antibody HGN194 was isolated from an individual infected with an HIV-1 clade AG recombinant circulating recombinant form (CRF). HGN194 targets an epitope in the third hypervariable loop (V3) of HIV-1 gp120 and neutralizes a range of relatively neutralization- sensitive and resistant viruses. We evaluated the potential of HGN194 to protect infant rhesus monkeys against a SHIV encoding a primary CCR5-tropic HIV-1 clade C envelope. After high- dose mucosal challenge, all untreated controls became highly viremic while all HGN194-treated animals (50 mg/kg) were completely protected. When HGN194 was given at 1 mg/kg, one out of two monkeys remained aviremic, whereas the other had delayed, lower peak viremia. Interestingly, all protected monkeys given high-dose HGN194 developed Gag-specific proliferative responses of both CD4+ and CD8+ T cells. To test whether generation of the latter involved cryptic infection, we ablated CD8+ cells after HGN194 clearance. No viremia was detected in any protected monkeys, thus ruling out virus reservoirs. Thus, induction of CD8 T-cell immunity may have resulted from transient “Hit and Run” infection or cross priming via Ag-Ab- mediated cross-presentation. Together, our data identified the HGN194 epitope as protective and provide proof-of-concept that this anti-V3 loop mAb can prevent infection with sterilizing immunity after challenge with virus of a different clade, implying that V3 is a potential vaccine target

Evaluation of cell-based and surrogate SARS-CoV-2 neutralization assays

Determinants of protective immunity against SARS-CoV-2 infection require the development of well-standardized, reproducible antibody assays. This need has led to the emergence of a variety of neutralization assays. Head-to-head evaluation of different SARS-CoV-2 neutralization platforms could facilitate comparisons across studies and laboratories. Five neutralization assays were compared using forty plasma samples from convalescent individuals with mild-to-moderate COVID-19: four cell-based systems using either live recombinant SARS-CoV-2 or pseudotyped viral particles created with lentivirus (LV) or vesicular stomatitis virus (VSV) packaging and one surrogate ELISA-based test that measures inhibition of the spike protein receptor binding domain (RBD) binding its receptor, human angiotensin converting enzyme 2 (hACE2). Vero, Vero E6, HEK293T expressing hACE2, and TZM-bl cells expressing hACE2 and transmembrane serine protease 2 were tested. All cell-based assays showed 50% neutralizing dilution (ND50) geometric mean titers (GMTs) that were highly correlated (Pearson r = 0.81–0.89) and ranged within 3.4-fold. The live-virus assay and LV-pseudovirus assays with HEK293T/hACE2 cells showed very similar mean titers: 141 and 178, respectively. ND50 titers positively correlated with plasma IgG targeting SARS-CoV-2 spike and RBD (r = 0.63–0.89), but moderately correlated with nucleoprotein IgG (r = 0.46–0.73). ND80 GMTs mirrored ND50 data and showed similar correlation between assays and with IgG concentrations. The VSV-pseudovirus assay and LV-pseudovirus assay with HEK293T/hACE2 cells in low and high-throughput versions were calibrated against the WHO SARS-CoV-2 IgG standard. High concordance between the outcomes of cell-based assays with live and pseudotyped virions enables valid cross-study comparison using these platforms. 24