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Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01–12) were isolated from donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (CDR) H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30–38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth and potency occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation. These data provide important insights relevant to HIV-1 vaccine development. A longitudinal study of an individual patient developing neutralizing antibodies against HIV-1 (targeting the V1V2 region of gp120) reveals how such neutralizing antibodies develop and evolve over time, providing important insights relevant to vaccine development. HIV-neutralizing antibody formation examined A better understanding of how HIV-1-neutralizing antibodies are generated could be a useful contribution to the design of improved AIDS vaccines. John Mascola and colleagues have now elucidated the immunological pathway of an important category of HIV-1-neutralizing antibody — those that target the variable V1V2 region of the viral envelope. These antibodies are more frequently elicited than CD4-binding site antibodies in the early stages of HIV infection and feature modest affinity maturation, a process that favours mutations in antibody variable domains that enhance antigen binding.

The identification of a new generation of potent broadly neutralizing HIV-1 antibodies (bnAbs) has generated substantial interest in their potential use for the prevention and/or treatment of HIV-1 infection. While combinations of bnAbs targeting distinct epitopes on the viral envelope (Env) will likely be required to overcome the extraordinary diversity of HIV-1, a key outstanding question is which bnAbs, and how many, will be needed to achieve optimal clinical benefit. We assessed the neutralizing activity of 15 bnAbs targeting four distinct epitopes of Env, including the CD4-binding site (CD4bs), the V1/V2-glycan region, the V3-glycan region, and the gp41 membrane proximal external region (MPER), against a panel of 200 acute/early clade C HIV-1 Env pseudoviruses. A mathematical model was developed that predicted neutralization by a subset of experimentally evaluated bnAb combinations with high accuracy. Using this model, we performed a comprehensive and systematic comparison of the predicted neutralizing activity of over 1,600 possible double, triple, and quadruple bnAb combinations. The most promising bnAb combinations were identified based not only on breadth and potency of neutralization, but also other relevant measures, such as the extent of complete neutralization and instantaneous inhibitory potential (IIP). By this set of criteria, triple and quadruple combinations of bnAbs were identified that were significantly more effective than the best double combinations, and further improved the probability of having multiple bnAbs simultaneously active against a given virus, a requirement that may be critical for countering escape in vivo. These results provide a rationale for advancing bnAb combinations with the best in vitro predictors of success into clinical trials for both the prevention and treatment of HIV-1 infection.

The aim of the study was to neutralize zearalenone by lactic acid bacteria (LAB) such as Lactococcus lactis and Bifidobacterium sp. and investigate the mechanism of zearalenone (ZEA) binding. Neutralization of ZEA by LAB was confirmed by identification of binding kinetics and spectroscopic studies such as Fourier transform infrared spectroscopy (FT-IR) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The obtained results showed that the kinetic process of zearalenone binding to L. lactis is not homogeneous but is expressed with an initial rapid stage with about 90% of ZEA biosorption and with a much slower second step. In case of Bifidobacterium sp., the neutralization process is homogeneous; the main stage can be described with about 88% of ZEA biosorption. MALDI–TOF-MS measurements and FTIR analysis confirmed the uptake of zearalenone molecules by bacterial species. Moreover, the assessment of dead and live lactic acid bacteria cells after zearalenone treatment was performed using fluorescence microscopy.

Abstract Background It is unknown whether a positive serology result correlates with protective immunity against SARS-CoV-2. There are also concerns regarding the low positive predictive value of SARS-CoV-2 serology tests, especially when testing populations with low disease prevalence. Methods A neutralization assay was validated in a set of PCR-confirmed positive specimens and in a negative cohort. In addition, 9530 specimens were screened using the Diazyme SARS-CoV-2 IgG serology assay and all positive results (N = 164 individuals) were reanalyzed using the neutralization assay, the Roche total immunoglobin assay, and the Abbott IgG assay. The relationship between the magnitude of a positive SARS-CoV-2 serology result and neutralizing activity was determined. Neutralizing antibody titers (50% inhibitory dilution, ID50) were also longitudinally monitored in patients confirmed to have SARS-CoV-2 by PCR. Results The SARS-CoV-2 neutralization assay had a positive percentage agreement (PPA) of 96.6% with a SARS-CoV-2 PCR test and a negative percentage agreement (NPA) of 98.0% across 100 negative control individuals. ID50 neutralization titers positively correlated with all 3 clinical serology platforms. Longitudinal monitoring of hospitalized PCR-confirmed patients with COVID-19 demonstrated they made high neutralization titers against SARS-CoV-2. PPA between the Diazyme IgG assay alone and the neutralization assay was 50.6%, while combining the Diazyme IgG assay with either the Roche or Abbott platforms increased the PPA to 79.2 and 78.4%, respectively. Conclusions These 3 clinical serology assays positively correlate with SARS-CoV-2 neutralization activity observed in patients with COVID-19. All patients confirmed SARS-CoV-2 positive by PCR develop neutralizing antibodies.

The CD4 binding site (CD4bs) of the HIV-1 envelope glycoprotein is susceptible to multiple lineages of broadly neutralizing antibodies (bnAbs) that are attractive to elicit with vaccines. The CH235 lineage (VH1-46) of CD4bs bnAbs is particularly attractive because the most mature members neutralize 90% of circulating strains, do not possess long HCDR3 regions, and do not contain insertions and deletions that may be difficult to induce. We used virus neutralization to measure the interaction of CH235 unmutated common ancestor (CH235 UCA) with functional Env trimers on infectious virions to guide immunogen design for this bnAb lineage. Two Env mutations were identified, one in loop D (N279K) and another in V5 (G458Y), that acted synergistically to render autologous CH505 transmitted/founder virus susceptible to neutralization by CH235 UCA. Man5-enriched N-glycans provided additional synergy for neutralization. CH235 UCA bound with nanomolar affinity to corresponding soluble native-like Env trimers as candidate immunogens. A cryo-EM structure of CH235 UCA bound to Man5-enriched CH505.N279K.G458Y.SOSIP.664 revealed interactions of the antibody light chain complementarity determining region 3 (CDR L3) with the engineered Env loops D and V5. These results demonstrate that virus neutralization can directly inform vaccine design and suggest a germline targeting and reverse engineering strategy to initiate and mature the CH235 bnAb lineage.

The development of a universal vaccine against foot-and-mouth disease virus (FMDV) is hindered by cross-serotype antigenic diversity and by a lack of knowledge regarding neutralization of the virus in natural hosts. In this study, we isolated serotype O-specific neutralizing antibodies (NAbs) (F145 and B77) from recovered natural bovine hosts by using the single B cell antibody isolation technique. We also identified a serotype O/A cross-reacting NAb (R50) and determined virus-NAb complex structures by cryo-electron microscopy at near-atomic resolution. F145 and B77 were shown to engage the capsid of FMDV-O near the icosahedral threefold axis, binding to the BC/HI-loop of VP2. In contrast, R50 engages the capsids of both FMDV-O and FMDV-A between the 2- and 5-fold axes and binds to the BC/EF/GH-loop of VP1 and to the GH-loop of VP3 from two adjacent protomers, revealing a previously unknown antigenic site. The cross-serotype neutralizing epitope recognized by R50 is highly conserved among serotype O/A. These findings help to elucidate FMDV neutralization by natural hosts and provide epitope information for the development of a universal vaccine for cross-serotype protection against FMDV.

Naja sumatrana and Naja kaouthia are medically important elapids species found in Southeast Asia. Snake bite envenoming caused by these species may lead to morbidity or mortality if not treated with the appropriate antivenom. In this study, the in vitro neurotoxic and myotoxic effects N . sumatrana and N . kaouthia venoms from Malaysian specimens were assessed and compared. In addition, the neutralizing capability of Cobra Antivenom (CAV), King Cobra Antivenom (KCAV) and Neuro Polyvalent Antivenom (NPAV) from Thailand were compared. Both venoms produced concentration-dependent neurotoxic and myotoxic effects in the chick biventer cervicis nerve-muscle preparation. Based on the time to cause 90% inhibition of twitches (i.e. t 90 ) N . kaouthia venom displayed more potent neurotoxic and myotoxic effects than N . sumatrana venom. All three of the antivenoms significantly attenuated venom-induced twitch reduction of indirectly stimulated tissues when added prior to venom. When added after N . sumatrana venom, at the t 90 time point, CAV and NPAV partially restored the twitch height but has no significant effect on the reduction in twitch height caused by N . kaouthia venom. The addition of KCAV, at the t 90 time point, did not reverse the attenuation of indirectly stimulated twitches caused by either venom. In addition, none of the antivenoms, when added prior to venom, prevented attenuation of directly stimulated twitches. Differences in the capability of antivenoms, especially NPAV and CAV, to reverse neurotoxicity and myotoxicity indicate that there is a need to isolate and characterize neurotoxins and myotoxins from Malaysian N . kaouthia and N . sumatrana venoms to improve neutralization capability of the antivenoms.

Snakebite envenomation inflicts a high burden of mortality and morbidity in sub-Saharan Africa. Antivenoms are the mainstay in the therapy of envenomation, and there is an urgent need to develop antivenoms of broad neutralizing efficacy for this region. The venoms used as immunogens to manufacture snake antivenoms are normally selected considering their medical importance and availability. Additionally, their ability to induce antibody responses with high neutralizing capability should be considered, an issue that involves the immunization scheme and the animal species being immunized. Using the lethality neutralization assay in mice, we compared the intrageneric neutralization scope of antisera generated by immunization of horses with monospecific, bispecific/monogeneric, and polyspecific/monogeneric immunogens formulated with venoms of Bitis spp., Echis spp., Dendroaspis spp., spitting Naja spp. or non-spitting Naja spp. It was found that the antisera raised by all the immunogens were able to neutralize the homologous venoms and, with a single exception, the heterologous congeneric venoms (considering spitting and non-spitting Naja separately). In general, the polyspecific antisera of Bitis spp, Echis spp, and Dendroaspis spp gave the best neutralization profile against venoms of these genera. For spitting Naja venoms, there were no significant differences in the neutralizing ability between monospecific, bispecific and polyspecific antisera. A similar result was obtained in the case of non-spitting Naja venoms, except that polyspecific antiserum was more effective against the venoms of N. melanoleuca and N. nivea as compared to the monospecific antiserum. The use of polyspecific immunogens is the best alternative to produce monogeneric antivenoms with wide neutralizing coverage against venoms of sub-Saharan African snakes of the Bitis, Echis, Naja (non-spitting) and Dendroaspis genera. On the other hand, a monospecific immunogen composed of venom of Naja nigricollis is suitable to produce a monogeneric antivenom with wide neutralizing coverage against venoms of spitting Naja spp. These findings can be used in the design of antivenoms of wide neutralizing scope for sub-Saharan Africa.

The role of C-X-C motif chemokine 10 (CXCL10), a pro-inflammatory factor, in the development of acute respiratory distress syndrome (ARDS) remains unclear. In this study, we explored the role of CXCL10 and the effect of CXCL10 neutralization in lipopolysaccharide (LPS)-induced ARDS in rats. The expression of CXCL10 and its receptor chemokine receptor 3(CXCR3) increased after LPS induction. Moreover, neutralization of CXCL10 ameliorated the severity of ARDS by reducing pulmonary edema, inhibiting the release of inflammatory mediators (IFN-γ, IL-6 and ICAM-1) and limiting inflammatory cells (neutrophils, macrophages, CD8+ T cells) influx into the lung, with a reduction in CXCR3 expression in neutrophils and macrophages. Therefore, CXCL10 could be a potential therapeutic target in LPS-induced ARDS.

Angiotensin-converting enzyme 2 (ACE2) is the main cellular receptor for the dangerous sarbecoviruses SARS-CoV and SARS-CoV-2. Its recombinant extracellular domain is used to monitor the level of protective humoral immune response to a viral infection or vaccine using the surrogate virus neutralization test (sVNT). Soluble ACE2 is also considered as an option for antiviral therapy potentially insensitive to the changes in the SARS-CoV-2 spike protein. Extensive testing of the samples of patient’s serum by the sVNT method requires using preparations of ACE2 or ACE2 conjugates with constant properties. We have previously obtained a cell line that is a producer of a soluble monomeric ACE2 and showed that this ACE2 variant can be used in sVNT, preferably as a conjugate with horseradish peroxidase. A cell line that generates an ACE2-Fc fusion protein with high productivity, more than 150 mg/liter of the target protein when cultured in a stirred flask, was obtained for producing a stable and universally applicable form of soluble ACE2. The affinity-purified ACE2-Fc fusion contains a mixture of dimeric and tetrameric forms, but allows obtaining linear response curves for inhibition of binding with the receptor-binding domain of the SARS-CoV-2 spike protein by antibodies. The ACE2-Fc-HRP-based sVNT testing system can be used for practical measurements of the levels of virus-neutralizing antibodies against various circulating variants of the SARS-CoV-2 virus.