Anti-idiotype isolation of a broad and potent influenza A virus-neutralizing human antibody.

The VH6-1 class of antibodies includes some of the broadest and most potent antibodies that neutralize influenza A virus. Here, we elicit and isolate anti-idiotype antibodies against germline versions of VH6-1 antibodies, use these to sort human leukocytes, and isolate a new VH6-1-class member, antibody L5A7, which potently neutralized diverse group 1 and group 2 influenza A strains. While its heavy chain derived from the canonical IGHV6-1 heavy chain gene used by the class, L5A7 utilized a light chain gene, IGKV1-9, which had not been previously observed in other VH6-1-class antibodies. The cryo-EM structure of L5A7 in complex with Indonesia 2005 hemagglutinin revealed a nearly identical binding mode to other VH6-1-class members. The structure of L5A7 bound to the isolating anti-idiotype antibody, 28H6E11, revealed a shared surface for binding anti-idiotype and hemagglutinin that included two critical L5A7 regions: an FG motif in the third heavy chain-complementary determining region (CDR H3) and the CDR L1 loop. Surprisingly, the chemistries of L5A7 interactions with hemagglutinin and with anti-idiotype were substantially different. Overall, we demonstrate anti-idiotype-based isolation of a broad and potent influenza A virus-neutralizing antibody, revealing that anti-idiotypic selection of antibodies can involve features other than chemical mimicry of the target antigen.

Expression and characterization of scFv-6009FV in Pichia pastoris with improved ability to neutralize the neurotoxin Cn2 from Centruroides noxius.

Small single-chain variable fragments (scFv) are promising biomolecules to inhibit and neutralize toxins and to act as antivenoms. In this work, we aimed to produce a functional scFv-6009FV in the yeast Pichia pastoris, which inhibits the pure Cn2 neurotoxin and the whole venom of Centruroides noxius. We were able to achieve yields of up to 31.6 ± 2 mg/L in flasks. Furthermore, the protein showed a structure of 6.1 % α-helix, 49.1 % ß-sheet, and 44.8 % of random coil by CD. Mass spectrometry confirmed the amino acid sequence and showed no glycosylation profile for this molecule. Purified scFv-6009FV allowed us to develop anti-scFvs in rabbits, which were then used in affinity columns to purify other scFvs. Determination of its half-maximal inhibitory concentration value (IC50) was 40 % better than the scFvs produced by E. coli as a control. Finally, we found that scFv-6009FV was able to inhibit ex vivo the pure Cn2 toxin and the whole venom from C. noxius in murine rescue experiments. These results demonstrated that under the conditions assayed here, P. pastoris is suited to produce scFv-6009FV that, compared to scFvs produced by E. coli, maintains the characteristics of an antibody and neutralizes the Cn2 toxin more effectively.

Gold-silver alloy hollow nanoshells-based lateral flow immunoassay for colorimetric, photothermal, and SERS tri-mode detection of SARS-CoV-2 neutralizing antibody.

Although many approaches have been developed for the quick assessment of SARS-CoV-2 infection, few of them are devoted to the detection of the neutralizing antibody, which is essential for assessing the effectiveness of vaccines. Herein, we developed a tri-mode lateral flow immunoassay (LFIA) platform based on gold-silver alloy hollow nanoshells (Au-Ag HNSs) for the sensitive and accurate quantification of neutralizing antibodies. By tuning the shell-to-core ratio, the surface plasmon resonance (SPR) absorption band of the Au-Ag HNSs is located within the near infrared (NIR) region, endowing them with an excellent photothermal effect under the irradiation of optical maser at 808 nm. Further, the Raman reporter molecule 4-mercaptobenzoic acid (MBA) was immobilized on the gold-silver alloy nanoshell to obtain an enhanced SERS signal. Thus, these Au-Ag HNSs could provide colorimetric, photothermal and SERS signals, with which, tri-mode strips for SARS-CoV-2 neutralizing antibody detection were constructed by competitive immunoassay. Since these three kinds of signals could complement one another, a more accurate detection was achieved. The tri-mode LFIA achieved a quantitative detection with detection limit of 20 ng/mL. Moreover, it also successfully detected the serum samples from 98 vaccinated volunteers with 79 positive results, exhibiting great application value in neutralizing antibody detection.

Protective anti-gB neutralizing antibodies targeting two vulnerable sites for EBV-cell membrane fusion.

Epstein-Barr virus (EBV) infects more than 90% of the world's adult population and accounts for a significant cancer burden of epithelial and B cell origins. Glycoprotein B (gB) is the primary fusogen essential for EBV entry into host cells. Here, we isolated two EBV gB-specific neutralizing antibodies, 3A3 and 3A5; both effectively neutralized the dual-tropic EBV infection of B and epithelial cells. In humanized mice, both antibodies showed effective protection from EBV-induced lymphoproliferative disorders. Cryoelectron microscopy analyses identified that 3A3 and 3A5 bind to nonoverlapping sites on domains D-II and D-IV, respectively. Structure-based mutagenesis revealed that 3A3 and 3A5 inhibit membrane fusion through different mechanisms involving the interference with gB-cell interaction and gB activation. Importantly, the 3A3 and 3A5 epitopes are major targets of protective gB-specific neutralizing antibodies elicited by natural EBV infection in humans, providing potential targets for antiviral therapies and vaccines.

Structural basis for ultrapotent antibody-mediated neutralization of human metapneumovirus.

Human metapneumovirus (hMPV) is a leading cause of morbidity and hospitalization among children worldwide, however, no vaccines or therapeutics are currently available for hMPV disease prevention and treatment. The hMPV fusion (F) protein is the sole target of neutralizing antibodies. To map the immunodominant epitopes on the hMPV F protein, we isolated a panel of human monoclonal antibodies (mAbs), and the mAbs were assessed for binding avidity, neutralization potency, and epitope specificity. We found the majority of the mAbs target diverse epitopes on the hMPV F protein, and we discovered multiple mAb binding approaches for antigenic site III. The most potent mAb, MPV467, which had picomolar potency, was examined in prophylactic and therapeutic mouse challenge studies, and MPV467 limited virus replication in mouse lungs when administered 24 h before or 72 h after viral infection. We determined the structure of MPV467 in complex with the hMPV F protein using cryo-electron microscopy to a resolution of 3.3 Å, which revealed a complex novel prefusion-specific epitope overlapping antigenic sites II and V on a single protomer. Overall, our data reveal insights into the immunodominant antigenic epitopes on the hMPV F protein, identify a mAb therapy for hMPV F disease prevention and treatment, and provide the discovery of a prefusion-specific epitope on the hMPV F protein.

Enhancing neutralization of Plasmodium falciparum using a novel monoclonal antibody against the rhoptry-associated membrane antigen.

The pathogenesis of malaria is associated with blood-stage infection and there is strong evidence that antibodies specific to parasite blood-stage antigens can control parasitemia. This provides a strong rational for applying blood-stage antigen components in a multivalent vaccine, as the induced antibodies in combination can enhance protection. The Plasmodium falciparum rhoptry-associated membrane antigen (PfRAMA) is a promising vaccine target, due to its fundamental role in merozoite invasion and low level of polymorphism. Polyclonal antibodies against PfRAMA are able to inhibit P. falciparum growth and interact synergistically when combined with antibodies against P. falciparum reticulocyte-binding protein 5 (PfRh5) or cysteine-rich protective antigen (PfCyRPA). In this study, we identified a novel PfRAMA-specific mAb with neutralizing activity, which in combination with PfRh5- or PfCyRPA-specific mAbs potentiated the neutralizing effect. By applying phage display technology, we mapped the protective epitope to be in the C-terminal region of PfRAMA. Our results confirmed previous finding of synergy between PfRAMA-, PfRh5- and PfCyRPA-specific antibodies, thereby paving the way of testing these antigens (or fragments of these antigens) in combination to improve the efficacy of blood-stage malaria vaccines. The results emphasize the importance of directing antibody responses towards protective epitopes, as the majority of anti-PfRAMA mAbs were unable to inhibit merozoite invasion of erythrocytes.

Latex Microsphere-Based Bicolor Immunochromatography for Qualitative Detection of Neutralizing Antibody against SARS-CoV-2.

Neutralizing antibody (NAb) is a family of antibodies with special functions, which afford a degree of protection against infection and/or reduce the risk of clinically severe infection. Receptor binding domain (RBD) in the spike protein of SARS-CoV-2, a portion of the S1 subunit, can stimulate the immune system to produce NAb after infection and vaccination. The detection of NAb against SARS-CoV-2 is a simple and direct approach for evaluating a vaccine's effectiveness. In this study, a direct, rapid, and point-of-care bicolor lateral flow immunoassay (LFIA) was developed for NAb against SARS-CoV-2 detection without sample pretreatment, and which was based on the principle of NAb-mediated blockage of the interaction between RBD and angiotensin-converting enzyme 2. In the bicolor LFIA, red and blue latex microspheres (LMs) were used to locate the test and control lines, leading to avoidance of erroneous interpretations of one-colored line results. Under the optimal conditions, NAb against SARS-CoV-2 detection carried out using the bicolor LFIA could be completed within 9 min, and the visible limit of detection was about 48 ng/mL. Thirteen serum samples were analyzed, and the results showed that the NAb levels in three positive serum samples were equal to, or higher than, 736 ng/mL. The LM-based bicolor LFIA allows one-step, rapid, convenient, inexpensive, and user-friendly determination of NAb against SARS-CoV-2 in serum.

Neutralizing antibodies induced in immunized macaques recognize the CD4-binding site on an occluded-open HIV-1 envelope trimer.

Broadly-neutralizing antibodies (bNAbs) against HIV-1 Env can protect from infection. We characterize Ab1303 and Ab1573, heterologously-neutralizing CD4-binding site (CD4bs) antibodies, isolated from sequentially-immunized macaques. Ab1303/Ab1573 binding is observed only when Env trimers are not constrained in the closed, prefusion conformation. Fab-Env cryo-EM structures show that both antibodies recognize the CD4bs on Env trimer with an 'occluded-open' conformation between closed, as targeted by bNAbs, and fully-open, as recognized by CD4. The occluded-open Env trimer conformation includes outwardly-rotated gp120 subunits, but unlike CD4-bound Envs, does not exhibit V1V2 displacement, 4-stranded gp120 bridging sheet, or co-receptor binding site exposure. Inter-protomer distances within trimers measured by double electron-electron resonance spectroscopy suggest an equilibrium between occluded-open and closed Env conformations, consistent with Ab1303/Ab1573 binding stabilizing an existing conformation. Studies of Ab1303/Ab1573 demonstrate that CD4bs neutralizing antibodies that bind open Env trimers can be raised by immunization, thereby informing immunogen design and antibody therapeutic efforts.

SARS-CoV-2 reactive and neutralizing antibodies discovered by single-cell sequencing of plasma cells and mammalian display.

Characterization of COVID-19 antibodies has largely focused on memory B cells; however, it is the antibody-secreting plasma cells that are directly responsible for the production of serum antibodies, which play a critical role in resolving SARS-CoV-2 infection. Little is known about the specificity of plasma cells, largely because plasma cells lack surface antibody expression, thereby complicating their screening. Here, we describe a technology pipeline that integrates single-cell antibody repertoire sequencing and mammalian display to interrogate the specificity of plasma cells from 16 convalescent patients. Single-cell sequencing allows us to profile antibody repertoire features and identify expanded clonal lineages. Mammalian display screening is used to reveal that 43 antibodies (of 132 candidates) derived from expanded plasma cell lineages are specific to SARS-CoV-2 antigens, including antibodies with high affinity to the SARS-CoV-2 receptor-binding domain (RBD) that exhibit potent neutralization and broad binding to the RBD of SARS-CoV-2 variants (of concern/interest).

Detection and quantification of antibody to SARS CoV 2 receptor binding domain provides enhanced sensitivity, specificity and utility.

Accurate and sensitive detection of antibody to SARS-CoV-2 remains an essential component of the pandemic response. Measuring antibody that predicts neutralising activity and the vaccine response is an absolute requirement for laboratory-based confirmatory and reference activity. The viral receptor binding domain (RBD) constitutes the prime target antigen for neutralising antibody. A double antigen binding assay (DABA), providing the most sensitive format has been exploited in a novel hybrid manner employing a solid-phase S1 preferentially presenting RBD, coupled with a labelled RBD conjugate, used in a two-step sequential assay for detection and measurement of antibody to RBD (anti-RBD). This class and species neutral assay showed a specificity of 100 % on 825 pre COVID-19 samples and a potential sensitivity of 99.6 % on 276 recovery samples, predicting quantitatively the presence of neutralising antibody determined by pseudo-type neutralization and by plaque reduction. Anti-RBD is also measurable in ferrets immunised with ChadOx1 nCoV-19 vaccine and in humans immunised with both AstraZeneca and Pfizer vaccines. This assay detects anti-RBD at presentation with illness, demonstrates its elevation with disease severity, its sequel to asymptomatic infection and its persistence after the loss of antibody to the nucleoprotein (anti-NP). It also provides serological confirmation of prior infection and offers a secure measure for seroprevalence and studies of vaccine immunisation in human and animal populations. The hybrid DABA also displays the attributes necessary for the detection and quantification of anti-RBD to be used in clinical practice. An absence of detectable anti-RBD by this assay predicates the need for passive immune prophylaxis in at-risk patients.

Memory B cell repertoire from triple vaccinees against diverse SARS-CoV-2 variants.

Omicron (B.1.1.529), the most heavily mutated SARS-CoV-2 variant so far, is highly resistant to neutralizing antibodies, raising concerns about the effectiveness of antibody therapies and vaccines1,2. Here we examined whether sera from individuals who received two or three doses of inactivated SARS-CoV-2 vaccine could neutralize authentic Omicron. The seroconversion rates of neutralizing antibodies were 3.3% (2 out of 60) and 95% (57 out of 60) for individuals who had received 2 and 3 doses of vaccine, respectively. For recipients of three vaccine doses, the geometric mean neutralization antibody titre for Omicron was 16.5-fold lower than for the ancestral virus (254). We isolated 323 human monoclonal antibodies derived from memory B cells in triple vaccinees, half of which recognized the receptor-binding domain, and showed that a subset (24 out of 163) potently neutralized all SARS-CoV-2 variants of concern, including Omicron. Therapeutic treatments with representative broadly neutralizing monoclonal antibodies were highly protective against infection of mice with SARS-CoV-2 Beta (B.1.351) and Omicron. Atomic structures of the Omicron spike protein in complex with three classes of antibodies that were active against all five variants of concern defined the binding and neutralizing determinants and revealed a key antibody escape site, G446S, that confers greater resistance to a class of antibodies that bind on the right shoulder of the receptor-binding domain by altering local conformation at the binding interface. Our results rationalize the use of three-dose immunization regimens and suggest that the fundamental epitopes revealed by these broadly ultrapotent antibodies are rational targets for a universal sarbecovirus vaccine.

A cell-free high throughput assay for assessment of SARS-CoV-2 neutralizing antibodies.

Highly accurate serological tests are key to assessing the prevalence of SARS-CoV-2 antibodies and the level of immunity in the population. This is important to predict the current and future status of the pandemic. With the recent emergence of new and more infectious SARS-CoV-2 variants, assays allowing for high throughput analysis of antibodies able to neutralize SARS-CoV-2 become even more important. Here, we report the development and validation of a robust, high throughput method, which enables the assessment of antibodies inhibiting the binding between the SARS-CoV-2 spike protein and angiotensin converting enzyme 2 (ACE2). The assay uses recombinantly produced spike-f and ACE2 and is performed in a bead array format, which allows analysis of up to 384 samples in parallel per instrument over seven hours, demanding only one hour of manual handling. The method is compared to a microneutralization assay utilising live SARS-CoV-2 and is shown to deliver highly correlating data. Further, a comparison with a serological method that measures all antibodies recognizing the spike protein shows that this type of assessment provides important insights into the neutralizing efficiency of the antibodies, especially for individuals with low antibody levels. This method can be an important and valuable tool for large-scale assessment of antibody-based neutralization, including neutralization of new spike variants that might emerge.

Development of a Purified Viral Preparation for Studies of COVID-19 (SARS-CoV-2) Biology.

Different methods for producing bulk quantities of concentrated and purified SARS-CoV-2 for the use as antigens and for the research into COVID-19 biology were tested.

A competitive ligand-binding assay to detect neutralizing antibodies to a bispecific drug using a multiplex Meso Scale Discovery platform.

Background: Monitoring appearance of neutralizing antibodies (NAbs) to multidomain large molecule drugs is a challenging task. Materials & methods: Here, we report development of a competitive ligand-binding assay for detection of NAbs to a bispecific candidate drug using a multiplex Meso Scale Discovery platform, which allows for detection of NAbs to both drug arms in the same sample. Results: The assay has sensitivity better than 250 ng/ml and is tolerant to the presence of drug at concentration >600 µg/ml and to the level of soluble target(s) >400 ng/ml. Conclusion: Our data suggest that multiplex approach can be successfully used for development of NAb assays in competitive ligand-binding assay format.

Comparative performance of multiplex salivary and commercially available serologic assays to detect SARS-CoV-2 IgG and neutralization titers.

Oral fluid (hereafter saliva) offers a non-invasive sampling method for detection of SARS-CoV-2 antibodies. However, data comparing performance of salivary tests against commercially-available serologic and neutralizing antibody (nAb) assays are lacking. This study compared the performance of a laboratory-developed multiplex salivary SARS-CoV-2 IgG assay targeting antibodies to nucleocapsid (N), receptor binding domain (RBD) and spike (S) antigens to three commercially-available SARS-CoV-2 serologic enzyme immunoassays (EIAs) (Ortho Vitros, Euroimmun, and BioRad) and nAb. Paired saliva and plasma samples were collected from 101 eligible COVID-19 convalescent plasma (CCP) donors >14 days since PCR+ confirmed diagnosis. Concordance was evaluated using positive (PPA) and negative (NPA) percent agreement, and Cohen's kappa coefficient. The range between salivary and plasma EIAs for SARS-CoV-2-specific N was PPA: 54.4-92.1% and NPA: 69.2-91.7%, for RBD was PPA: 89.9-100% and NPA: 50.0-84.6%, and for S was PPA: 50.6-96.6% and NPA: 50.0-100%. Compared to a plasma nAb assay, the multiplex salivary assay PPA ranged from 62.3% (N) and 98.6% (RBD) and NPA ranged from 18.8% (RBD) to 96.9% (S). Combinations of N, RBD, and S and a summary algorithmic index of all three (N/RBD/S) in saliva produced ranges of PPA: 87.6-98.9% and NPA: 50-91.7% with the three EIAs and ranges of PPA: 88.4-98.6% and NPA: 21.9-34.4% with the nAb assay. A multiplex salivary SARS-CoV-2 IgG assay demonstrated variable, but comparable performance to three commercially-available plasma EIAs and a nAb assay, and may be a viable alternative to assist in monitoring population-based seroprevalence and vaccine antibody response.

Hybrid immunity improves B cells and antibodies against SARS-CoV-2 variants.

The emergence of SARS-CoV-2 variants is jeopardizing the effectiveness of current vaccines and limiting the application of monoclonal antibody-based therapy for COVID-19 (refs. 1,2). Here we analysed the memory B cells of five naive and five convalescent people vaccinated with the BNT162b2 mRNA vaccine to investigate the nature of the B cell and antibody response at the single-cell level. Almost 6,000 cells were sorted, over 3,000 cells produced monoclonal antibodies against the spike protein and more than 400 cells neutralized the original SARS-CoV-2 virus first identified in Wuhan, China. The B.1.351 (Beta) and B.1.1.248 (Gamma) variants escaped almost 70% of these antibodies, while a much smaller portion was impacted by the B.1.1.7 (Alpha) and B.1.617.2 (Delta) variants. The overall loss of neutralization was always significantly higher in the antibodies from naive people. In part, this was due to the IGHV2-5;IGHJ4-1 germline, which was found only in people who were convalescent and generated potent and broadly neutralizing antibodies. Our data suggest that people who are seropositive following infection or primary vaccination will produce antibodies with increased potency and breadth and will be able to better control emerging SARS-CoV-2 variants.

Versatile and rapid microfluidics-assisted antibody discovery.

Recent years have seen unparalleled development of microfluidic applications for antibody discovery in both academic and pharmaceutical research. Microfluidics can support native chain-paired library generation as well as direct screening of antibody secreting cells obtained by rodent immunization or from the human peripheral blood. While broad diversities of neutralizing antibodies against infectious diseases such as HIV, Ebola, or COVID-19 have been identified from convalescent individuals, microfluidics can expedite therapeutic antibody discovery for cancer or immunological disease indications. In this study, a commercially available microfluidic device, Cyto-Mine, was used for the rapid identification of natively paired antibodies from rodents or human donors screened for specific binding to recombinant antigens, for direct screening with cells expressing the target of interest, and, to our knowledge for the first time, for direct broad functional IgG antibody screening in droplets. The process time from cell preparation to confirmed recombinant antibodies was four weeks. Application of this or similar microfluidic devices and methodologies can accelerate and enhance pharmaceutical antibody hit discovery.

Broad betacoronavirus neutralization by a stem helix-specific human antibody.

The spillovers of betacoronaviruses in humans and the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants highlight the need for broad coronavirus countermeasures. We describe five monoclonal antibodies (mAbs) cross-reacting with the stem helix of multiple betacoronavirus spike glycoproteins isolated from COVID-19 convalescent individuals. Using structural and functional studies, we show that the mAb with the greatest breadth (S2P6) neutralizes pseudotyped viruses from three different subgenera through the inhibition of membrane fusion, and we delineate the molecular basis for its cross-reactivity. S2P6 reduces viral burden in hamsters challenged with SARS-CoV-2 through viral neutralization and Fc-mediated effector functions. Stem helix antibodies are rare, oftentimes of narrow specificity, and can acquire neutralization breadth through somatic mutations. These data provide a framework for structure-guided design of pan-betacoronavirus vaccines eliciting broad protection.

Nanobodies as powerful pulmonary targeted biotherapeutics against SARS-CoV-2, pharmaceutical point of view.

Background Since December 2019, the newly emerged SARS-CoV-2 virus continues to infect humans and many people died from severe Covid-19 during the last 2 years worldwide. Different approaches are being used for treatment of this infection and its consequences, but limited results have been achieved and new therapeutics are still needed. One of the most interesting biotherapeutics in this era are Nanobodies which have shown very promising results in recent researches. Scope of review Here, we have reviewed the potentials of Nanobodies in Covid-19 treatment. We have also discussed the properties of these biotherapeutics that make them very suitable for pulmonary drug delivery, which seems to be very important route of administration in this disease. Major conclusion Nanobodies with their special biological and biophysical characteristics and their resistance against harsh manufacturing condition, can be considered as promising, targeted biotherapeutics which can be administered by pulmonary delivery pharmaceutical systems against Covid-19. General significance Covid-19 has become a global problem during the last two years and with emerging mutant strains, prophylactic and therapeutic approaches are still highly needed. Nanobodies with their specific properties can be considered as valuable and promising candidates in Covid-19 therapy.