anti-HIV Antibodies Research Articles

The influence of proline isomerization on potency and stability of anti-HIV antibody 10E8

Monoclonal antibody (mAb) 10E8 recognizes a highly conserved epitope on HIV and is capable of neutralizing > 95% of circulating viral isolates making it one of the most promising Abs against HIV. Solution instability and biochemical heterogeneity of 10E8 has hampered its development for clinical use. We identify the source of 10E8 heterogeneity being linked to cis/trans isomerization at two prolines within the YPP motif in the CRD3 loop that exists as two predominant conformers that interconvert on a slow timescale. The YtransP conformation conformer can bind the HIV gp41 epitope, while the YcisP is not binding competent and shows a higher aggregation propensity. The high barrier of isomerization and propensity to adopt non-binding competent proline conformers provides novel insight into the slow binding kinetics, low potency, and poor solubility of 10E8. This study highlights how proline isomerization should be considered a critical quality attribute for biotherapeutics with paratopes containing potential cis proline amide bonds.

Exploring optical spectroscopic techniques and nanomaterials for virus detection.

Viral infections pose significant health challenges globally by affecting millions of people worldwide and consequently resulting in a negative impact on both socioeconomic development and health. Corona virus disease 2019 (COVID-19) is a clear example of how a virus can have a global impact in the society and has demonstrated the limitations of detection and diagnostic capabilities globally. Another virus which has posed serious threats to world health is the human immunodeficiency virus (HIV) which is a lentivirus of the retroviridae family responsible for causing acquired immunodeficiency syndrome (AIDS). Even though there has been a significant progress in the HIV biosensing over the past years, there is still a great need for the development of point of care (POC) biosensors that are affordable, robust, portable, easy to use and sensitive enough to provide accurate results to enable clinical decision making. The aim of this study was to present a proof of concept for detecting HIV-1 pseudoviruses by using anti-HIV1 gp41 antibodies as capturing antibodies. In our study, glass substrates were treated with a uniform layer of silane in order to immobilize HIV gp41 antibodies on their surfaces. Thereafter, the HIV pseudovirus was added to the treated substrates followed by addition of anti-HIV gp41 antibodies conjugated to selenium nanoparticle (SeNPs) and gold nanoclusters (AuNCs). The conjugation of SeNPs and AuNCs to anti-HIV gp41 antibodies was characterized using UV–vis spectroscopy, transmission electron microscopy (TEM) and zeta potential while the surface morphology was characterized by fluorescence microscopy, atomic force microscopy (AFM) and Raman spectroscopy. The UV–vis and zeta potential results showed that there was successful conjugation of SeNPs and AuNCs to anti-HIV gp41 antibodies and fluorescence microscopy showed that antibodies immobilized on glass substrates were able to capture intact HIV pseudoviruses. Furthermore, AFM also confirmed the capturing HIV pseudoviruses and we were able to differentiate between substrates with and without the HIV pseudoviruses. Raman spectroscopy confirmed the presence of biomolecules related to HIV and therefore this system has potential in HIV biosensing applications.

Epidemiology of HIV, Hepatitis B and C virus in emergency and elective surgical patients: Our experience of 275 patients in a single surgical ward

Human immunodeficiency, hepatitis B and hepatitis C virus are major health problems throughout the world. All over the world surgeons have the highest risk for exposure to blood and its products during performing their procedures. Human immunodeficiency, hepatitis B and hepatitis C affecting more than three billion people worldwide and hepatitis B and hepatitis C virus is the single most important factor of chronic liver disease and hepato-cellular carcinoma in India and abroad. Universally preoperative testing of these blood born virus HIV, HBV, HCV has been a accepted strategy to reduce the risk of virus transmission. This study was designed to analyse the magnitude of these infections as a global health problem, affecting millions of population throughout globe. This study also describe the prevalence of Human Immunodeficiency Virus, Hepatitis B and Hepatitis C virus in surgical patients undergoing elective and emergency surgeries. Material and Methods: This is a retrospective study done on 275 patients who were admitted for general surgical care between July 2016 and May 2020 in single surgical ward and were positive for HBsAg, anti-HCV, and anti-HIV antibodies. Results: Patients included in the study were between 16-18 years of age and included both male as well as female. There were 182 male and 93 female with male: female ratio 1: 0.51. Out of 275 patients 33.45% patients were positive for HBsAg, 34.54% were positive for anti-HCV, and 29.09% were positive for anti-HIV. Conclusions: Education of all healthcare workers including doctors along with routine preoperative testing of all patients undergoing elective and emergency surgeries are highly important to enhance the awareness of these infection in order to reduce the transmission of disease. As the common message is ‘Prevention is better than cure ‘It is highly important to prevent further spread of these infections by screening of every patient before any surgery whether elective or emergency and also counselling should be done of all patients. All the doctors and paramedical staff must follow the proper ethical practice to ensuring the use of sterile disposables in all these patients where indicated and also protecting themselves from these viral infections.

P1122VIRAL INFECTIONS :VHC, VHB AND VIH IN HEMODIALYSIS

Abstract Background and Aims Viral infections, especially those caused by hepatitis B and C viruses are common in chronic hemodialysis patients. The hepatitis C virus is the main cause of post-transfusion hepatitis. The main objective of this study is to determine the prevalence of hepatitis B and C and HIV in chronic hemodialysis patients at the TIZI OUZOU University Hospital Center, as well as the main risk factors for contamination, in chronic hemodialysis patients treated at the from our center. Method This retrospective study carried out on 149 chronic hemodialysis patients consisted in searching for anti-HCV, anti-HIV antibodies and the HBs antigen and in determining the possible causes of contamination, in particular the age of the hemodialysis and history of blood transfusion. The characteristics studied for all patients are: demographic characteristics, initial nephropathy, seniority on hemodialysis, the concept of hemodialysis outside our center at the time of dialysis or for a limited period during follow-up ( vacation…), the notion of transfusion; patients are considered to be multi-transfused when they have received more than four transfusions, the search for hepatitis B and C virus infection by ELISA, the concept of hepatic cytolysis: a transaminase level that exceeds 1.5 times normal, vaccine status, HIV serology Results The average age of our 149 patients varies between 19 and 82 years with a male predominance (92 men and 57 women) and an average duration of dialysis of 90 months. The predominant etiology of IRCT is vascular nephropathy. We also note other causes including glomerular, diabetic and tubulointerstitial nephropathies. The etiology was largely undetermined. Hepatic serology C is positive in 5% of patients. None of our patients tested positive for HBS. No case of HIV infection has been noted. The prevalence of hepatitis B and C in hemodialysis patients varies from country to country but remains higher than in the general population (HCV: 2.7-22.2% in the DOPPS register). In our series the prevalence is around 5% lower than that reported in other regional and international studies. Conclusion In hemodialysis, the risk of infection is very high, on the one hand due to access to regular blood and on the other hand due to the alteration of the immune defenses induced by chronic renal failure. Nosocomial transmission plays a very important role. It is strongly linked to the age of dialysis. Compliance with hygiene measures is a very essential element; isolation of the infected patient is no longer recommended

Reduction of anti-HIV antibody responses in subjects receiving antiretroviral therapy during chronic HIV-1 infection.

Antiretroviral therapy (ART) can lead to a decline or absence of anti-HIV antibodies in HIV-infected children or acutely HIV-infected (AHI) subjects. However, the characteristics of anti-HIV antibody response in the subjects who are treated during chronic HIV-1 infection (CHI) have not yet been fully investigated. Different anti-HIV antibodies were longitudinally quantified and analyzed in 81 CHI adults under ART. The factors associated with antibody decline were evaluated by binary logistic regression analysis. ART led to 36.0% (27/75) and 52.1% (38/73) of the patients whose anti-HIV levels reduced by more than 75% of the baseline levels at 12 and 24 months post-ART, respectively. The reduction of anti-HIV antibodies correlated with the decline of HIV-1 viral load with correlation coefficients in the range 0.556-0.848 or R2 value of 0.576-0.873 (P < 0.001). However, no negative detection of anti-HIV antibody was observed at 24 months post-ART. The time from HIV-1 diagnosis to ART initiation and the baseline anti-HIV levels were the key factors associated with quick decline of anti-HIV antibodies during ART. ART-induced kinetics of anti-HIV antibody response was different among the subjects with AHI and CHI. Misdiagnosis of HIV-1 infection may not be a serious issue in HIV-1 chronically infected subjects under ART, and could ideally be avoided by using multiple HIV-1 antigens for screening purposes.

Plant-based production of highly potent anti-HIV antibodies with engineered posttranslational modifications

Broadly neutralising antibodies (bNAbs) against human immunodeficiency virus type 1 (HIV-1), such as CAP256-VRC26 are being developed for HIV prevention and treatment. These Abs carry a unique but crucial post-translational modification (PTM), namely O-sulfated tyrosine in the heavy chain complementarity determining region (CDR) H3 loop. Several studies have demonstrated that plants are suitable hosts for the generation of highly active anti-HIV-1 antibodies with the potential to engineer PTMs. Here we report the expression and characterisation of CAP256-VRC26 bNAbs with posttranslational modifications (PTM). Two variants, CAP256-VRC26 (08 and 09) were expressed in glycoengineered Nicotiana benthamiana plants. By in planta co-expression of tyrosyl protein sulfotransferase 1, we installed O-sulfated tyrosine in CDR H3 of both bNAbs. These exhibited similar structural folding to the mammalian cell produced bNAbs, but non-sulfated versions showed loss of neutralisation breadth and potency. In contrast, tyrosine sulfated versions displayed equivalent neutralising activity to mammalian produced antibodies retaining exceptional potency against some subtype C viruses. Together, the data demonstrate the enormous potential of plant-based systems for multiple posttranslational engineering and production of fully active bNAbs for application in passive immunisation or as an alternative for current HIV/AIDS antiretroviral therapy regimens.

An efficient method for simultaneously screening for HIV, syphilis, and HCV based on one dried blood spot sample

ObjectiveThe aim of this study was to optimize the parameters of a dried blood spot (DBS)-based ELISA method to simultaneously screen for anti-HIV, anti-hepatitis C virus (HCV) and anti-treponema pallidum (TP) antibodies, and investigate the assay performance. MethodsExperiments were performed to establish optimized parameters for a DBS-based ELISA method to simultaneously screen for anti-HIV, anti-HCV, and anti-TP antibodies. Then, 429 paired plasma and DBS samples were collected to evaluate the performance of the assay with optimized parameters. Plasma test results were used as the reference standard. ResultsThe optimized assay conditions were: blood volume, 70–100 μL; DBS size, whole spot; eluent volume, 500 μL; eluent, PBS with 1‰ Tween20; elution time, 4 h; elution temperature, room temperature. The overall prevalence of HIV, HCV, and TP was 6.06% (95% confidence interval [CI]: 4.07–8.87%), 27.74% (95% CI: 23.60–32.28%), and 14.92% (95% CI: 11.75–18.73%). The clinical sensitivity of the assay for anti-HIV, anti-HCV, and anti-TP antibodies was 88.46%, 98.32%, and 92.19%, respectively. The assay was 100% specificity for each analyte. The assay positive predictive value for each analyte was 100%, and the negative predictive values were >98%. Of the 429 samples, 9 DBS results were different than the plasma results; in these samples the plasma signal-to-cutoff rations were low (range, 1.40–7.81). ConclusionThe DBS-based ELISA method demonstrated good performance for simultaneously screening for anti-HIV, anti-HCV, and anti-TP antibodies. DBS samples are a promising method to screen for HIV, HCV, and TP infections. ImportanceDBS samples are a promising alternative to plasma samples, with the advantages of good sample stability, smaller blood volume, simpler storage, and easier transport. DBS sample testing is used for the diagnosis of a wide variety of pathogens, including HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV). The performance of DBS samples for the serological diagnosis of HIV, HCV, and Treponema pallidum (TP) has been demonstrated to be good in many studies, and therefore DBS is regarded as an ideal choice to screen for HIV, HCV, and TP infections, especially in difficult-access or resource-limited settings. There is no generally accepted procedure for sample collection and processing of DBS samples and there are few studies on simultaneous detection of anti-HIV, anti-HCV and anti-TP using a single DBS.

Response to Zhao and Zhou: Diagnosis of HIV infection in breastfed infants of mothers on antiretroviral therapy.

We thank Zhao and Zhou [1] for their thoughtful comments on our report of two infants born to women with HIV who had unusual trajectories of diagnostic tests. The two HIV-exposed infants were initially positive on nucleic acid amplification tests in the first month of life but had multiple negative tests during breastfeeding and presented with definitive evidence of HIV infection around the time of breastfeeding cessation. We hypothesize that constituents of breast milk, or processes induced by breastfeeding, most likely in combination with antiviral effects of maternal antiretroviral therapy, led to the undetectable test results that we observed in already infected infants until breastfeeding ended. We considered the alternative explanation of postnatal transmission proposed by Zhao and Zhou [1], but this explanation requires dismissing the initial positive test results on the early samples as transient infections or false-positive results. A rigorous investigation of 42 cases of suspected transient infection, failed to confirm a single one, despite the plausibility of this concept [2]. In one of the infants in our report, virus was detected in peripheral blood mononuclear cells (PBMCs) from three separate blood samples collected over the first month of life making laboratory errors an unlikely explanation. In this same case, CD4+ T cells from one of these early age time points were found to be positive for 2-LTR episomal HIV-1 DNA. Detection of 2-LTR episomal DNA in CD4+ T cells, although at low levels, provides evidence of viral replication as these episomal circles are a by-product thereof [3]. We acknowledge that early samples were not available for the second case making the alternative explanation of an initial false-positive result followed by postnatal infection a possible explanation of this second case. It should be noted that current birth testing and early treatment guidelines in South Africa would provide indefinite treatment to transient infections should they occur [4]. We would like to clarify the implications of our observations in the context of current guidance around antiretroviral use for women and their children to prevent vertical transmission of HIV. Maintenance of maternal therapeutic antiretroviral drug regimens over the full duration of breastfeeding and beyond is now considered standard of care for prevention of HIV transmission to children. This recommendation is consistent with clinical trials demonstrating exceptionally low rates of postnatal transmission if effective maternal antiretroviral drug treatment is in place [5]. Longer term infant prophylaxis is recommended only for infants whose mothers initiated treatment late and/or who are nonadherent with treatment. In both cases, maternal viral loads through the breastfeeding period were less than 1000 RNA copies/ml and in-country treatment guidelines were followed in terms of prophylactic regimens [6]. Because of concerns that infant prophylaxis may mask underlying HIV infections, some have encouraged repeat testing after prophylaxis has ended [7]. What distinguishes our cases is that the pattern of negative results was observed after discontinuation of antiretrovirals given to the infants but during continuation of maternal antiretroviral treatment and breastfeeding. Although the profile of the results that we observed is rare, we support repeat testing of HIV-exposed breastfed infants after complete cessation of all breastfeeding. The profile of HIV antibodies is of great interest, but it is unlikely that changes in standard anti-HIV IgG antibody titres over time in these infants will be helpful to resolve the timing of transmission. Infant antiretroviral prophylaxis. and on-going antiretroviral exposure via breast milk, like early treatment, may lead to undetectable antibodies [8]. The ability to detect HIV antibody would probably be at the time when these factors are no longer in place and viral levels rise and drive antibody production. Thus, a rise in antibodies, only once breastfeeding has stopped, might not necessarily mean the children were infected later. More sensitive assays differentiating antibody isotypes and antigen specificity may be more informative but will need comparison with the usual profile of antibodies in HIV-exposed uninfected children. The utility of HIV antibodies as markers of viral processes is well appreciated and this is likely to be a productive, albeit complex, line of research. Acknowledgements Conflicts of interest There are no conflicts of interest.

HIV antibodies level as a marker of HIV persistence: the role of hepatitis C virus coinfection.

Human immunodeficiency virus (HIV) antibodies have been proposed as a measure of the size of the HIV reservoir. The aim of our study is to quantify the anti-HIV antibodies level in a cohort of people living with HIV (PLWH), stratified based on the presence of continuous undetectable HIV viral load and the co-existence of hepatitis C virus infection. A sample of 229 HIV-monoinfected (n = 114) or HIV/HCV-coinfected [either with resolved HCV infection (n = 75) or active HCV coinfection (n = 40)] patients, followed up a median of 34 (IQR 20-44) months, was studied. Anti-HIV index was obtained as the 1:800 dilution of HIV antibodies. CD4+ T cell count, time with undetectable HIV viral load, annual increase of CD4+ T cell count, anti-HCV therapy, and diagnosis of cirrhosis were analyzed. Patients with a continued suppressed HIV viral load had significant lower anti-HIV index compared with those with virologic failure during the follow-up. Significant higher CD4+ T cell increase was observed in those with a lower anti-HIV index. HIV-monoinfected patients showed an anti-HIV index significantly lower than patients with HCV coinfection. Resolved HCV infection after interferon-based therapy, but not with direct acting antivirals, was associated with a lower anti-HIV index. HIV/HCV-coinfected patients showed higher HIV antibodies level when compared with HIV-monoinfected individuals. A decrease in anti-HIV index in HIV/HCV-coinfected patients was detected when a sustained virological HCV response was obtained after interferon-based therapy, in possible relation with the direct or indirect effect of interferon on PLWH CD4 T cells.

Triple infection with Cryptococcus, varicella-zoster virus, and Mycobacterium abscessus in a patient with anti-interferon-gamma autoantibodies: a case report

BackgroundThe most common infection in patients positive for anti-interferon-gamma autoantibodies (anti-IFN-γ AAbs) is disseminated nontuberculous mycobacterial (dNTM) infection. Here, we report a rare case of triple infection caused by Cryptococcus, varicella-zoster virus (VZV), and nontuberculous mycobacterium in a patient with anti-IFN-γ AAbs.Case presentationA 53-year-old Thai man presented with a progressively enlarging right cervical mass with low-grade fever and significant weight loss for 4 months. He also developed a lesion at his left index finger. A biopsy of that lesion showed granulomatous inflammation with yeast-like organisms morphologically consistent with cryptococcosis. Serum cryptococcal antigen was positive. Histopathology of a right cervical lymph node revealed chronic granulomatous lymphadenitis, and the lymph node culture grew Mycobacterium abscessus. One month later, he complained of vision loss in his left eye and subsequently developed a group of painful vesicles at the right popliteal area of S1 dermatome. Lumbar puncture was performed and his cerebrospinal fluid was positive for VZV DNA. His blood test for anti-HIV antibody was negative. Anti-IFN-γ AAbs was positive, but test for anti-granulocyte-macrophage colony-stimulating factor autoantibodies (anti-GM-CSF AAbs) was negative. He was treated with amphotericin B plus fluconazole for cryptococcosis; a combination of amikacin, imipenem, azithromycin, and levofloxacin for dNTM infection; and, intravenous acyclovir for disseminated VZV infection. After treatment, our patient’s fever and cervical lymphadenopathy were subsided, and his vision and visual acuity were both improved.ConclusionsThis is the first case of triple infection with cryptococcosis, VZV, and dNTM in a patient who tested positive for anti-IFN-γ AAbs and negative for anti-GM-CSF AAbs. This case will increase awareness and heighten suspicion of these infections in patients with the described presentations and clinical characteristics, and this will accelerate diagnosis and treatment.

Long-Term Delivery of an Anti-SIV Monoclonal Antibody With AAV.

Long-term delivery of anti-HIV monoclonal antibodies using adeno-associated virus (AAV) holds promise for the prevention and treatment of HIV infection. We previously reported that after receiving a single administration of AAV vector coding for anti-SIV antibody 5L7, monkey 84-05 achieved high levels of AAV-delivered 5L7 IgG1 in vivo which conferred sterile protection against six successive, escalating dose, intravenous challenges with highly infectious, highly pathogenic SIVmac239, including a final challenge with 10 animal infectious doses (1). Here we report that monkey 84-05 has successfully maintained 240–350 μg/ml of anti-SIV antibody 5L7 for over 6 years. Approximately 2% of the circulating IgG in this monkey is this one monoclonal antibody. This monkey generated little or no anti-drug antibodies (ADA) to the AAV-delivered antibody for the duration of the study. Due to the nature of the high-dose challenge used and in order to rule out a potential low-level infection not detected by regular viral loads, we have used ultrasensitive techniques to detect cell-associated viral DNA and RNA in PBMCs from this animal. In addition, we have tested serum from 84-05 by ELISA against overlapping peptides spanning the whole envelope sequence for SIVmac239 (PepScan) and against recombinant p27 and gp41 proteins. No reactivity has been detected in the ELISAs indicating the absence of naturally arising anti-SIV antibodies; moreover, the ultrasensitive cell-associated viral tests yielded no positive reaction. We conclude that macaque 84-05 was effectively protected and remained uninfected. Our data show that durable, continuous antibody expression can be achieved after one single administration of AAV and support the potential for lifelong protection against HIV from a single vector administration.

Mathematical modeling to reveal breakthrough mechanisms in the HIV Antibody Mediated Prevention (AMP) trials.

The ongoing Antibody Mediated Prevention (AMP) trials will uncover whether passive infusion of the broadly neutralizing antibody (bNAb) VRC01 can protect against HIV acquisition. Previous statistical simulations indicate these trials may be partially protective. In that case, it will be crucial to identify the mechanism of breakthrough infections. To that end, we developed a mathematical modeling framework to simulate the AMP trials and infer the breakthrough mechanisms using measurable trial outcomes. This framework combines viral dynamics with antibody pharmacokinetics and pharmacodynamics, and will be generally applicable to forthcoming bNAb prevention trials. We fit our model to human viral load data (RV217). Then, we incorporated VRC01 neutralization using serum pharmacokinetics (HVTN 104) and in vitro pharmacodynamics (LANL CATNAP database). We systematically explored trial outcomes by reducing in vivo potency and varying the distribution of sensitivity to VRC01 in circulating strains. We found trial outcomes could be used in a clinical trial regression model (CTRM) to reveal whether partially protective trials were caused by large fractions of VRC01-resistant (IC50>50 μg/mL) circulating strains or rather a global reduction in VRC01 potency against all strains. The former mechanism suggests the need to enhance neutralizing antibody breadth; the latter suggests the need to enhance VRC01 delivery and/or in vivo binding. We will apply the clinical trial regression model to data from the completed trials to help optimize future approaches for passive delivery of anti-HIV neutralizing antibodies.

Higher Baseline ADCC Responses in Chronic Nonprogressive HIV Infection Are Associated with Reduced HIV Burden in Later Course of Disease.

The importance of anti-HIV antibodies mediating antibody-dependent cell-mediated cytotoxicity (ADCC) in protective immunity against HIV is recognized recently. The purpose of this study was to measure the functional ADCC response at different stages of HIV infection in a well-defined HIV+ cohort, including 20 recently infected individuals, 30 with long-term slow-progressive, 24 with short-term slow-progressive and 32 with progressive HIV infection using a rapid fluorometric ADCC assay. The antibodies mediating ADCC were found in all disease stages. These antibodies were detectable at as early as 25 days after the estimated date of infection, however, did not influence the viral load set point probably indicating no major influence on the early course of the disease. However, the frequency and magnitude of functional ADCC responses were associated with higher CD4+T cell count and lower viral load and were significantly lower in progressors compared with other groups. The usefulness of the ADCC responses in longer viral control was assessed in a subset of participants with slowly progressing HIV infection. In these individuals, the ADCC responses observed at the visit 1 were found to be increased over time and were associated with lower plasma viral load estimated 4 to 15 years later in the disease course. Overall, the study findings confirm the role of ADCC antibodies in reducing the viral burden and also indicate the probable role of sustained functional ADCC responses in reducing the viral burden during the later period of HIV infection.

Effects of a remote mutation from the contact paratope on the structure of CDR-H3 in the anti-HIV neutralizing antibody PG16

PG16 is a broadly neutralizing antibody to the human immunodeficiency virus (HIV). A crystal structure of PG16 revealed that the unusually long 28-residue complementarity determining region (CDR) H3 forms a unique subdomain, referred to as a “hammerhead”, that directly contacts the antigen. The hammerhead apparently governs the function of PG16 while a previous experimental assay showed that the mutation of TyrH100Q to Ala, which does not directly contact the antigen, decreased the neutralization ability of PG16. However, the molecular mechanism by which a remote mutation from the hammerhead or contact paratope affects the neutralization potency has remained unclear. Here, we performed molecular dynamics simulations of the wild-type and variants (TyrH100Q to Ala, and TyrH100Q to Phe) of PG16, to clarify the effects of these mutations on the dynamics of CDR-H3. Our simulations revealed that the structural rigidity of the CDR-H3 in PG16 is attributable to the hydrogen bond interaction between TyrH100Q and ProH99, as well as the steric support by TyrH100Q. The loss of both interactions increases the intrinsic fluctuations of the CDR-H3 in PG16, leading to a conformational transition of CDR-H3 toward an inactive state.

OP 8.2 - How long is long-term? Delivery of anti-HIV antibodies using AAV vector

OP 8.2 - How long is long-term? Delivery of anti-HIV antibodies using AAV vector

Liver-Directed but Not Muscle-Directed AAV-Antibody Gene Transfer Limits Humoral Immune Responses in Rhesus Monkeys

A number of publications have described the use of adeno-associated virus (AAV) for the delivery of anti-HIV and anti-simian immunodeficiency virus (SIV) monoclonal antibodies (mAbs) to rhesus monkeys. Anti-drug antibodies (ADAs) have been frequently observed, and long-term AAV-mediated delivery has been inconsistent. Here, we investigated different AAV vector strategies and delivery schemes to rhesus monkeys using the rhesus monkey mAb 4L6. We compared 4L6 immunoglobulin G1 (IgG1) delivery using the AAV1 versus the AAV8 serotype with a cytomegalovirus (CMV) promoter and the use of a muscle-specific versus a liver-specific promoter. Long-term expression levels of 4L6 IgG1 following AAV8-mediated gene transfer were comparable to those following AAV1-mediated gene transfer. AAV1-mediated gene transfer, using a muscle-specific promoter, showed robust ADAs and transiently low 4L6 IgG1 levels that ultimately declined to below detectable levels. Intravenous AAV8-mediated gene transfer, using a liver-specific promoter, also resulted in low levels of delivered 4L6 IgG1, but those low levels were maintained in the absence of any detectable ADAs. Booster injections using AAV1-CMV allowed for increased 4L6 IgG1 serum levels in animals that were primed with AAV8 but not with AAV1. Our results suggest that liver-directed expression may help to limit ADAs and that re-administration of AAV of a different serotype can result in successful long-term delivery of an immunogenic antibody.

Identification of functional mimetics of anti-hiv antibody n6 by methods of virtual screening and molecular modeling

Three chemical compounds presenting functional mimetics of neutralizing anti-HIV-1 antibody N6 were identified by an integrated approach including virtual screening, high-throughput docking and molecular dynamics. Using molecular docking, the identified compounds are predicted to be able to block three key regions of the HIV-1 gp120 protein by formation of a wide network of intermolecular contacts with the glycoprotein residues critical for the virus binding to primary receptor CD4. It is shown that the complexes of these compounds with gp120 exhibit low values of dissociation constant and Gibbs free energy, which validates a high efficacy of intermolecular interactions stabilizing these supramolecular structures. Based on the data obtained, the identified compounds are assumed to form promising basic structures for the development of novel, potent and broad anti-HIV-1 drugs mimicking structural and functional properties of the cross-reactive neutralizing antibody N6.

2516. Persistence of Anti-HIV Antibodies in HIV-1-infected Patients on Combination Antiretroviral Therapy (cART) with Prolonged Viral Suppression

BackgroundDespite years of cART with sustained HIV-RNA plasma viral load (pVL) < 50 copies/mL, antibodies (Abs) to HIV-1 proteins persist. The reasons for this are unknown but may reflect long-lived B cell responses and/or persistence of antigen. To address which of these may be the case, we compared decay rates of anti-HIV Abs to those of anti-Measles Abs.MethodsA cohort of 10 HIV-infected patients on cART with pVL < 50 (average 3 years, range 0–7.1 years) were studied. Baseline and 5-year follow-up serum were collected and assessed for anti-HIV-1 Abs via western blot (Cambridge Biotech WB kit), with additional densiometric analysis used for quantitative calculation of a western blot “score” that was normalized to control specimens. The kinetics of measles Abs titers over the same 5-year period were also analyzed by the quantitative Serion Measles IgG ELISA kit, given a history of prior vaccination in these participants. McNemar’s and paired t-tests were used for analysis.ResultsAll 10 patients exhibited persistence of anti-HIV-1 Abs at ≥ 5 year follow-up as determined by persistence of ≥ 2 diagnostic bands on WB (P = 0.003). The patterns for all 10 participants varied individually by patient with each exhibiting a unique profile (representative figure); 7/10 patients had WBs that could be analyzed quantitatively and showed no significant change over the study period (average baseline score 5.9 vs 5.3 at follow-up, P = 0.26). 7/9 patients had valid measles IgG measurements showing a consistent but nonsignificant decline over the same 5-year study period (measles titer 55–2800 mIU/mL at baseline vs 50–1300 mIU/mL at follow-up, n = 9, P = 0.18).ConclusionThe mechanism leading to the persistence of anti-HIV-1 Abs in HIV-infected individuals with prolonged viral suppression has been unclear. The general decline in anti-Measles IgG titer over the 5-year observational period in 7/9 patients is likely to be explained by waning B cell-mediated humoral immunity in these vaccinated individuals and consistent with their lack of ongoing exposure to this pathogen. The persistence of anti-HIV-1 Abs over the same 5-year period, on the other hand, may indicate persistent HIV-1 viral protein production either from active reservoirs or by transcription of “defective” HIV-1 proviruses as recently reported. Disclosures All authors: No reported disclosures.

A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli

Monoclonal antibodies (mABs) are of great biopharmaceutical importance for the diagnosis and treatment of diseases. However, their production in mammalian expression hosts usually requires extensive production times and is expensive. Escherichia coli has become a new platform for production of functional small antibody fragment variants. In this study, we have used a rhamnose-inducible expression system that allows precise control of protein expression levels. The system was first evaluated for the cytoplasmic production of super folder green fluorescence protein (sfGFP) in various production platforms and then for the periplasmic production of the anti-HIV single-chain variable antibody fragment (scFv) of PGT135. Anti-HIV broadly neutralizing antibodies, like PGT135, have potential for clinical use to prevent HIV transmission, to promote immune responses and to eradicate infected cells. Different concentrations of L-rhamnose resulted in the controlled production of both sfGFP and scFv PGT135 antibody. In addition, by optimizing the culture conditions, the amount of scFv PGT135 antibody that was expressed soluble or as inclusions bodies could be modulated. The proteins were produced in batch bioreactors, with yields of 4.9 g/L for sfGFP and 0.8 g/L for scFv. The functionality of the purified antibodies was demonstrated by their ability to neutralize a panel of different HIV variants in vitro. We expect that this expression system will prove very useful for the development of a more cost-effective production process for proteins and antibody fragments in microbial cells.

Epidemiology, clinical features, and associated factors in 78 cases of lichen planus on black skin.

The pathogenesis of lichen planus (LP) is mostly autoimmune, while psychological and infectious factors are recognized to trigger or aggravate the disease. An association with diabetes is reported. Our objective was to determine the epidemio-clinical characteristics of LP and its associated factors. This multicentric, prospective study was conducted over a 6-month period. The histopathology was only performed for atypical forms. Patients with a notion of drug intake before the rash were excluded. Anti-hepatitis C Virus (HCV) antibodies screening was systematical in case of mucosal damage. The data were analyzed using the SPSS IBM 20 software. The average age was 38years. Women represented 84.6% (n=66) of the studied population. The patients were married in 61.5%. Obesity or overweight status was noted in 41%. A marital or relational conflict was found in 25.6%. History of LP was reported in 24.4% (n=19). Pruritus was found in 96.2%. The locations were as follows: skin (97.4%), mucous membranes (15.4%), and hair and nails (5.1%). Lesions were diffuse in 56.4%. The clinical forms were as follows: typical (52.6%), erythematosquamous (17%), warty (14.5%), pigmented (14.5%), and blaschkolinear (one case). Histopathology confirmed the diagnosis of LP in 91.4%. Blood sugar level was high in one case. Hepatitis B surface antigen (HBsAg) was positive in 3.03%. Anti-HIV and anti-HCV antibodies were negative. Lichen planus is a relatively rare disease in sub-Saharan Africa and is seen more in adults. The clinical manifestations are polymorphic, but the mucosal damage is rarely isolated.