Antibodies with modest neutralizing activity and narrow breadth are commonly elicited in HIV-1. Here, we evaluated the complementary and synergistic activities of a set of monoclonal antibodies (MAb) isolated from a single patient, directed to V3, CD4 binding site (CD4bs), and CD4 induced (CD4i) epitopes. Despite low somatic hypermutation percentages in the variable regions, these MAbs covered viral strains from subtypes B, C, A and CRF01_AE and transmitted/founder viruses in terms of binding, neutralizing and antibody-dependent cell-mediated cytotoxicity (ADCC) activities. In addition, a combination of the anti-V3 and CD4bs MAbs showed a synergistic effect over the neutralization of HIV-1JR-FL. A humoral response from a single patient covered a wide range of viruses by complementary and synergistic activities of antibodies with different specificities. Inducing a set of narrow neutralizing antibodies, easier to induce than the broadly neutralizing antibodies, could be a strategy for developing an effective vaccine against HIV-1.

Specific transport systems for penicillins have been recognized, but their in vivo role in the context of other transporters remains unclear. We produced a serum against amoxicillin (anti-AMPC) conjugated to albumin with glutaraldehyde. The antiserum was specific for AMPC and ampicillin (ABPC) but crossreacted weakly with cephalexin. This enabled us to develop an immunocytochemical (ICC) method for detecting the uptake of AMPC in the rat intestine, liver, and kidney. Three hours after a single oral administration of AMPC, the ICC method revealed that AMPC distributed to a high degree in the microvilli, nuclei, and cytoplasm of the absorptive epithelial cells of the intestine. AMPC distributed in the cytoplasm and nuclei of the hepatocytes in a characteristic granular morphology on the bile capillaries, and in addition, AMPC adsorption was observed on the luminal surface of the capillaries, intercalated portions, and interlobular bile ducts on the bile flow. Almost no AMPC could be detected 6 h postadministration in either the intestine or the liver. Meanwhile, in the kidney, AMPC persisted until 12 h postadministration to a high degree in the proximal tubules, especially in the S3 segment cells in the tubular lumen, in which numerous small bodies that strongly reacted with the antibody were observed. All these sites of AMPC accumulation correspond well to specific sites where certain transporter systems for penicillins occur, suggesting that AMPC is actually and actively absorbed, eliminated, or excreted at these sites, possibly through such certain penicillin transporters.Amoxicillin (AMPC) is a moderate-spectrum, bacteriolytic, ␤-lactam antibiotic used to treat bacterial infections caused by susceptible microorganisms, acting by inhibiting the synthesis of the bacterial cell wall. In chemotherapy, the ability of a drug to reach its site of action for a desired duration is dependent on absorption, distribution, metabolism, and excretion, all of which are intimately related to the transport mechanisms in the barrier epithelia (56). Actually, pharmacotherapeutic efficacy and toxicity are governed in vivo by a multitude of pharmacodynamic and pharmacokinetic factors. Recently, a variety of transporters for penicillins have been demonstrated at the molecular level, especially in the kidney and liver, in which numerous potentially toxic xenobiotics and drugs are eliminated (23). It has been postulated that the following may be involved in the transport of penicillins: in the small intestine, the proton-coupled oligopeptide transporter PEPT1 (1); in the liver, the organic anion transporter (OAT) (5, 22, 44, 54), multidrug resistance-associated protein (Mrp2) (6,26,39,42), and sodium-dependent phosphate transport protein (NPT1) (8, 21, 57); and in the kidney, the rat multispecific organic anion transporter 1 (rOAT1), rOAT2, and rOAT3 (2, 22, 23, 51, 55), Mrp2 (43), and H ϩ /peptide cotransporters (PEPT1 and PEPT2) (23-25, 38, 45, 49, 53), etc. The interaction of such transporters with ␤-lactam antibiotics has been stu...

SUMMARY:The V3 loop in the envelope (Env) of HIV-1 is one of the major targets of neutralizing antibodies. However, this antigen is hidden inside the Env trimer in most isolates and is fully exposed only during CD4-gp120 interaction. Thus, primary HIV-1 isolates are relatively resistant to anti-V3 antibodies because IgG is too large to access the V3 loop. To overcome this obstacle, we constructed singlechain variable fragments (scFvs) from anti-V3 monoclonal antibodies 0.5g, 5G2, and 16G6. Enhanced neutralization by 0.5g and 5G2 scFvs was observed in strains resistant to their IgG counterparts. Neutralization coverage by 0.5g scFv reached up to 90z of the tested viruses (tier 2 and 3 classes). The temperature-regulated neutralization assay revealed that extensive cross-neutralization of 0.5g scFv can be explained by post-attachment neutralization. Neutralization assay involving viruses carrying an intersubunit disulfide bond (SOS virus) showed that the neutralization-susceptible timeframe after attachment was 60 to 120 min. These results indicate that the scFvs efficiently access the V3 loop and subsequently neutralize HIV-1, even after virus attachment to the target cells. Based on its broad and potent neutralizing activity, further development of anti-V3 scFv for therapeutic and preventive strategies is warranted.

BackgroundHIV-1 typically develops resistance to any single antiretroviral agent. Combined anti-retroviral therapy to reduce drug-resistance development is necessary to control HIV-1 infection. Here, to assess the utility of a combination of antibody and fusion inhibitor treatments, we investigated the potency of monoclonal antibodies at neutralizing HIV-1 variants that are resistant to fusion inhibitors.ResultsMutations that confer resistance to four fusion inhibitors, enfuvirtide, C34, SC34, and SC34EK, were introduced into the envelope of HIV-1JR-FL, a CCR5-tropic tier 2 strain. Pseudoviruses with these mutations were prepared and used for the assessment of neutralization sensitivity to an array of antibodies. The resulting neutralization data indicate that the potencies of some antibodies, especially of those against the CD4 binding site, V3 loop, and membrane-proximal external region epitopes, were increased by the mutations in gp41 that conferred resistance to the fusion inhibitors. C34-, SC34-, and SC34EK-resistant mutants showed more sensitivity to monoclonal antibodies than enfuvirtide-resistant mutants. An analysis of C34-resistant mutations revealed that the I37K mutation in gp41 HR1 is a key mutation for C34 resistance, low infectivity, neutralization sensitivity, epitope exposure, and slow fusion kinetics. The N126K mutation in the gp41 HR2 domain contributed to C34 resistance and neutralization sensitivity to anti-CD4 binding site antibodies. In the absence of L204I, the effect of N126K was antagonistic to that of I37K. The results of a molecular dynamic simulation of the envelope trimer confirmation suggest that an I37K mutation induces the augmentation of structural fluctuations prominently in the interface between gp41 and gp120. Our observations indicate that the “conformational unmasking” of envelope glycoprotein by an I37K mutation is one of the mechanisms of neutralization sensitivity enhancement. Furthermore, the enhanced neutralization of C34-resistant mutants in vivo was shown by its high rate of neutralization by IgG from HIV patient samples.ConclusionsMutations in gp41 that confer fusion inhibitor resistance exert enhanced sensitivity to broad neutralizing antibodies (e.g., VRC01 and 10E8) and other conventional antibodies developed in HIV-1 infected patients. Therefore, next-generation fusion inhibitors and monoclonal antibodies could be a potential combination for future regimens of combined antiretroviral therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0304-7) contains supplementary material, which is available to authorized users.

Pulsed discharge plasmas, a type of non-thermal plasma, have received much attention as an abundant radical source. Pulsed discharge is mainly divided into two phases: A streamer discharge, which has a streamer head with very high electron energy; and a glow-like discharge, which forms between electrodes uniformly. Many types of radical are produced by the streamer head, while the glow-like discharge produces many radicals due to high electron density. However, various physical characteristics of pulsed discharge such as time history of electron energy and electron temperature remain unclear.This work observes the propagation process and time history of electron energy for radial direction of pulsed discharge in a coaxial electrode using high-speed digital framing camera and emission spectroscopy. In the experiment, the pulse generator consists of a Blumlein line and a pulse transformer. The Blumlein line has 400ns of pulsed duration, and pulse transformer has a winding ratio of 1 to 3. The coaxial-type electrode has an inner diameter of 0.5mm, an outer diameter of 60mm, and a length of 10 mm. The feeding gas into the coaxial electrode was nitrogen in this experiment.Framing images show that streamer discharges were initiated at the inner electrode and terminated at the outer electrode. Therefore, the results were also able to observe the change from streamer discharge to glow-like discharge. This paper will report the propagation process and time history of pulsed discharge.