A Combination of M50I and V151I Polymorphic Mutations in HIV-1 Subtype B Integrase Results in Defects in Autoprocessing

We have recently reported that a recombinant HIV-1NL4.3 containing Met-to-Ile change at codon 50 of integrase (IN) (IN:M50I) exhibits suppression of the virus release below 0.5% of WT HIV, and the released viral particles are replication-incompetent due to defects in Gag/GagPol processing by inhibition of the initiation of autoprocessing of GagPol polyproteins in the virions and leads to replication-incompetent viruses. The coexisting Ser-to-Asn change at codon 17 of IN or Asn-to-Ser mutation at codon 79 of RNaseH (RH) compensated the defective IN:M50I phenotype, suggesting that both IN and RH regulate an HIV infectability. In the current study, to elucidate a distribution of the three mutations during anti-retroviral therapy among patients, we performed a population analysis using 529 plasma virus RNA sequences obtained through the MiSeq. The result demonstrated that 14 plasma HIVs contained IN:M50I without the compensatory mutations. Comparing the sequences of the 14 viruses with that of the defective virus illustrated that only Val-to-Ile change at codon 151 of IN (IN:V151I) existed in the recombinant virus. This IN:V151I is known as a polymorphic mutation and was derived from HIVNL4.3 backbone. A back-mutation at 151 from Ile-to-Val in the defective virus recovered HIV replication capability, and Western Blotting assay displayed that the back-mutation restored Gag/GagPol processing in viral particles. These results demonstrate that a combination of IN:M50I and IN:V151I mutations, but not IN:M50I alone, produces a defective virus.

Methylene blue photochemical treatment as a reliable SARS-CoV-2 plasma virus inactivation method for blood safety and convalescent plasma therapy for the COVID-19 outbreak

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Comparison of the coagulation potential of lyophilized blood plasma virus-inactivated by two different methods.

Введение. Свежезамороженная плазма (СЗП) — один из самых распространённых компонентов крови, применяемых сегодня в клиниках при оказании медицинской помощи при кровотечениях и тяжёлых коагулопатиях. В отличие от вирусинактивированной замороженной плазмы, сублимированная (лиофилизированная) плазма может храниться при комнатной температуре, и восстановление перед переливанием обычно требует меньших временных затрат. Цель исследования: оценить коагуляционный потенциал лиофилизированной плазмы, полученной из вирусинактивированной плазмы, инактивированной 2 способами: с использованием метиленового синего + видимый свет и рибофлавина + ультрафиолетовое облучение спектра B. Материалы и методы. Проведен анализ 100 образцов иофилизированной плазмы, вирусинактивированной двумя методами. Изучали влияние лиофилизации на уровень факторов свертывания и показатели свертываемости в вирусинактивированной плазме. Для сравнительной оценки в качестве контроля были проанализированы 150 образцов СЗП. Результаты. При использовании обоих технологий инактивации в лиофилизированной вирусинактивированной плазме установлено снижение содержания факторов V и VIII как по отношению к СПЗ, так и по отношению к физиологической норме. Лиофилизация вирусинактивированной плазмы различными методами привела к некоторому увеличению показателей свёртывания крови — протромбинового времени и активированного частичного тромбопластинового времени. Остальные показатели оставались в нормальных пределах. Существенных различий в показателях между образцами плазмы, инактивированной различными методами, выявлено не было. Заключение. По клиническим свойствам вирусинактивированная лиофилизированная плазма может служить альтернативой СЗП, однако для уточнения всесторонних аспектов её применения необходимы дополнительные исследования. Introduction. Fresh frozen plasma (FFP) is one of the most common blood components used today in clinics for medical care of bleeding and severe coagulopathies. Unlike virus-inactivated frozen plasma, sublimated (lyophilized) plasma can be stored at room temperature, and recovery before transfusion usually requires less time. Objectives: to assess the coagulation potential of lyophilized plasma obtained from virus- inactivated plasma inactivated by 2 methods: using methylene blue + visible light and riboflavin + ultraviolet radiation of spectrum B. Materials/Methods. Analysis of 100 samples of lyophilized plasma, virus-inactivated by 2 methods, was carried out. The effect of lyophilization on the level of coagulation factors and coagulation parameters in virus-inactivated plasma was studied. For comparative evaluation, 150 samples of FFP were analyzed as a control. Results. Using both technologies for inactivation of lyophilized virus- inactivated plasma, a decrease in the content of V and VIII factors was found both in relation to the FFP and in relation to the physiological norm. Lyophilization of virus-inactivated plasma by various methods led to a slight increasing in blood coagulation parameters — prothrombin time and activated partial thromboplastin time. The rest of the parameters remained within normal limits. There were no significant differences in parameters between plasma samples inactivated by different methods. Conclusions. According clinical properties, virus- inactivated lyophilized plasma can serve as an alternative to FFP, but more studies are needed to clarify the comprehensive aspects of its use.

HIV-1C env and gag Variation in the Cerebrospinal Fluid and Plasma of Patients with HIV-Associated Cryptococcal Meningitis in Botswana

HIV-1 compartmentalization in reservoir sites remains a barrier to complete HIV eradication. It is unclear whether there is variation in HIV-1 env and gag between cerebrospinal fluid (CSF) and plasma of individuals with HIV-associated cryptococcal meningitis (CM). We compared HIV-1 env characteristics and the gag cytotoxic T-lymphocyte (CTL) escape mutations from CSF and plasma samples. Employing population-based Sanger sequencing, we sequenced HIV-1 env from CSF of 25 patients and plasma of 26 patients. For gag, 15 CSF and 21 plasma samples were successfully sequenced. Of these, 18 and 9 were paired env and gag CSF/plasma samples, respectively. There was no statistically significant difference in the proportion of CCR5-using strains in the CSF and plasma, (p = 0.50). Discordant CSF/plasma virus co-receptor use was found in 2/18 pairs (11.1%). The polymorphisms in the HIV-1 V3 loop were concordant between the two compartments. From the HIV-1 gag sequences, three pairs had discordant CTL escape mutations in three different epitopes of the nine analyzed. These findings suggest little variation in the HIV-1 env between plasma and CSF and that the CCR5-using strains predominate in both compartments. HIV-1 gag CTL escape mutations also displayed little variation in CSF and plasma suggesting similar CTL selective pressure.

959. HIV-1 DNA Testing Identifies Drug Resistance in Viremic Patients with Pan-Sensitive Plasma Virus

Background HIV-1 drug resistance mutations (DRMs) are lost from plasma virus in the absence of selective drug pressure. Therefore, viremic patients who present with pan-sensitive plasma HIV-1 may harbor archived drug resistance that hinders virologic suppression upon reinstatement of therapy. We sought to determine if testing HIV-1 DNA in peripheral blood mononuclear cells (PBMCs) can identify drug resistance in patients with pan-sensitive plasma virus. Methods Sixty-four patients were identified who had HIV-1 RNA and HIV-1 DNA drug resistance testing performed on the same day and demonstrated pan-sensitive virus on the HIV-1 RNA test. Antiretroviral (ARV) resistance and DRMs identified by each test were compared between 66 test pairs. Patients were stratified by viral load (VL) to assess its impact on resistance detection using t-test and correlation analyses. Results The mean patient age was 37; 92% were female. Most (94%) were infected with HIV-1 subtype B, with an average VL of 110,000 c/mL (150 - 1,470,000 c/mL) at the time of resistance testing. Resistance to at least one ARV was reported on 20% (13/66) of HIV-1 DNA tests, and was associated with nucleos(t)ide and non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs), protease inhibitors (PIs), and integrase inhibitors (INIs) on 4.5%, 9%, 4.5%, and 6% of HIV-1 DNA reports, respectively. Across all HIV-1 DNA tests, 74 DRMs were identified that were not found in the plasma virus, including 12 NRTI, 16 NNRTI, 39 PI, and 7 INI DRMs; M184V was identified on 4.5% (3/66) of HIV-1 DNA reports. The mean VL was not significantly different between HIV-1 DNA tests reporting resistance to at least one ARV (101,000 c/mL) and those reporting pan-sensitivity (110,000 c/mL). Viral load at time of testing did not correlate with the number of ARVs to which resistance was reported. Conclusion In viremic patients with pan-sensitive plasma virus, HIV-1 DNA testing can identify drug resistance regardless of VL level. Assessment of drug resistance in HIV-1 DNA may be useful in designing suppressive ARV regimens for patients whose plasma virus DRMs fail to be identified due to lack of adherence or continuity of care. Disclosures Dusica Curanovic, PhD, Monogram Biosciences (Employee) Sharon K. Martens, MN, Monogram Biosciences (Employee) Milka A. Rodriguez, PhD, Monogram Biosciences (Employee) Christos J. Petropoulos, PhD, Monogram Biosciences (Employee) Charles M. Walworth, MD, Monogram Biosciences (Employee)

Fc-mediated effector function contributes to the in vivo antiviral effect of an HIV neutralizing antibody

Treatment of HIV infection with either antiretroviral (ARV) therapy or neutralizing monoclonal antibodies (NAbs) leads to a reduction in HIV plasma virus. Both ARVs and NAbs prevent new rounds of viral infection, but NAbs may have the additional capacity to accelerate the loss of virus-infected cells through Fc gamma receptor (FcγR)-mediated effector functions, which should affect the kinetics of plasma-virus decline. Here, we formally test the role of effector function in vivo by comparing the rate and timing of plasma-virus clearance in response to a single-dose treatment with either unmodified NAb or those with either reduced or augmented Fc function. When infused into viremic simian HIV (SHIV)-infected rhesus macaques, there was a 21% difference in slope of plasma-virus decline between NAb and NAb with reduced Fc function. NAb engineered to increase FcγRIII binding and improve antibody-dependent cellular cytotoxicity (ADCC) in vitro resulted in arming of effector cells in vivo, yet led to viral-decay kinetics similar to NAbs with reduced Fc function. These studies show that the predominant mechanism of antiviral activity of HIV NAbs is through inhibition of viral entry, but that Fc function can contribute to the overall antiviral activity, making them distinct from standard ARVs.

Comparison of the coagulation potential of lyophilized blood plasma virus-inactivated by various methods

The article presents research data on the conservation of coagulation potential of lyophilized plasma inactivated by three different technologies — amotosalen and ultraviolet irradiation of spectrum A, riboflavin + ultraviolet of spectrum B, methylene blue + visible light. The study analyzed the concentration of blood-coagulation factors that affect the extrinsic, intrinsic and general coagulation pathways by comparing samples of virus-inactivated lyophilized plasma with various inactivation methods. As a result of the study, no significant differences in the indices between samples of plasma inactivated by various methods were detected. Therefore, virus-inactivated lyophilized plasma can serve as a full alternative to fresh frozen plasma.

Methylene blue photochemical treatment as a reliable SARS-CoV-2 plasma virus inactivation method for blood safety and convalescent plasma therapy for the COVID-19 outbreak

Background With the outbreak of unknown pneumonia in Wuhan, China in December 2019, a new coronavirus (SARS-CoV-2) attracted worldwide attention. Although coronaviruses typically infect the upper or lower respiratory tract, discovery of the virus in plasma is common. Therefore, the risk of transmitting coronavirus through transfusion of blood products remains. As more asymptomatic infections are found in COVID-19 cases, blood safety is shown to be particularly important, especially in endemic areas. Study Design and MethodsBX-1, an ‘AIDS treatment instrument’ based on methylene blue (MB) photochemical technology, developed by Boxin (Beijing) Biotechnology Development LTD, has proven that inactivation of lipid-enveloped viruses such as HIV-1 in plasma has high efficiency, without damage to other components in the plasma, and proved safe and reliable in clinical trials of HIV treatment. In order to confirm the inactivation effect of BX-1 in SARS-CoV-2, we used the SARS-CoV-2 virus strain isolated from Zhejiang University for plasma virus inactivation studies. Results and ConclusionBX-1 can effectively eliminate SARS-CoV-2 within 2 mins, and the virus titer decline can reach 4.5 log10 TCID50/mL. Faced with the expanding epidemic, BX-1 is safe for blood transfusion and plasma transfusion therapy in recovery patients, and the inactivated vaccine preparation has great potential for treatment in the current outbreak.