Fusion peptide-directed antibodies: Humoral armor against HIV-1 infection.

Infused neutralizing antibodies to the fusion peptide of the HIV envelope glycoprotein protected macaques from mucosal viral challenge (Pegu et al.).

Glycan heterogeneity as a cause of the persistent fraction in HIV-1 neutralization.

Neutralizing antibodies (NAbs) to multiple epitopes on the HIV-1-envelope glycoprotein (Env) have been isolated from infected persons. The potency of NAbs is measured more often than the size of the persistent fraction of infectivity at maximum neutralization, which may also influence preventive efficacy of active or passive immunization and the therapeutic outcome of the latter. Many NAbs neutralize HIV-1 CZA97.012, a clone of a Clade-C isolate, to ~100%. But here NAb PGT151, directed to a fusion-peptide epitope, left a persistent fraction of 15%. NAb PGT145, ligating the Env-trimer apex, left no detectable persistent fraction. The divergence in persistent fractions was further analyzed by depletion of pseudoviral populations of the most PGT151- and PGT145-reactive virions. Thereby, neutralization by the non-depleting NAb increased, whereas neutralization by the depleting NAb decreased. Furthermore, depletion by PGT151 increased sensitivity to autologous neutralization by sera from rabbits immunized with soluble native-like CZA97.012 trimer: substantial persistent fractions were reduced. NAbs in these sera target epitopes comprising residue D411 at the V4-ß19 transition in a defect of the glycan shield on CZA97.012 Env. NAb binding to affinity-fractionated soluble native-like CZA97.012 trimer differed commensurately with neutralization in analyses by ELISA and surface plasmon resonance. Glycan differences between PGT151- and PGT145-purified trimer fractions were then demonstrated by mass spectrometry, providing one explanation for the differential antigenicity. These differences were interpreted in relation to a new structure at 3.4-Å resolution of the soluble CZA97.012 trimer determined by cryo-electron microscopy. The trimer adopted a closed conformation, refuting apex opening as the cause of reduced PGT145 binding to the PGT151-purified form. The evidence suggests that differences in binding and neutralization after trimer purification or pseudovirus depletion with PGT145 or PGT151 are caused by variation in glycosylation, and that some glycan variants affect antigenicity through direct effects on antibody contacts, whereas others act allosterically.

Conformational antigenic heterogeneity as a cause of the persistent fraction in HIV-1 neutralization.

BACKGROUND: Neutralizing antibodies (NAbs) protect against HIV-1 acquisition in animal models and show promise in treatment of infection. They act by binding to the viral envelope glycoprotein (Env), thereby blocking its receptor interactions and fusogenic function. The potency of neutralization is largely determined by affinity. Less well explained is the persistent fraction, the plateau of remaining infectivity at the highest antibody concentrations. RESULTS: We observed different persistent fractions for neutralization of pseudovirus derived from two Tier-2 isolates of HIV-1, BG505 (Clade A) and B41 (Clade B): it was pronounced for B41 but not BG505 neutralization by NAb PGT151, directed to the interface between the outer and transmembrane subunits of Env, and negligible for either virus by NAb PGT145 to an apical epitope. Autologous neutralization by poly- and monoclonal NAbs from rabbits immunized with soluble native-like B41 trimer also left substantial persistent fractions. These NAbs largely target a cluster of epitopes lining a hole in the dense glycan shield of Env around residue 289. We partially depleted B41-virion populations by incubating them with PGT145- or PGT151-conjugated beads. Each depletion reduced the sensitivity to the depleting NAb and enhanced it to the other. Autologous neutralization by the rabbit NAbs was decreased for PGT145-depleted and enhanced for PGT151-depleted B41 pseudovirus. Those changes in sensitivity encompassed both potency and the persistent fraction. We then compared soluble native-like BG505 and B41 Env trimers affinity-purified by each of three NAbs: 2G12, PGT145, or PGT151. Surface plasmon resonance showed differences among the fractions in antigenicity, including kinetics and stoichiometry, congruently with the differential neutralization. The large persistent fraction after PGT151 neutralization of B41 was attributable to low stoichiometry, which we explained structurally by clashes that the conformational plasticity of B41 Env causes. CONCLUSION: Distinct antigenic forms even of clonal HIV-1 Env, detectable among soluble native-like trimer molecules, are distributed over virions and may profoundly mold neutralization of certain isolates by certain NAbs. Affinity purifications with some antibodies may yield immunogens that preferentially expose epitopes for broadly active NAbs, shielding less cross-reactive ones. NAbs reactive with multiple conformers will together reduce the persistent fraction after passive and active immunization.

Conformational antigenic heterogeneity as a cause of the persistent fraction in HIV-1 neutralization.

Background Neutralizing antibodies (NAbs) protect against HIV-1 acquisition in animal models and show promise in treatment of infection. They act by binding to the viral envelope glycoprotein (Env), thereby blocking its receptor interactions and fusogenic function. The potency of neutralization is largely determined by affinity. Less well explained is the persistent fraction, the plateau of remaining infectivity at the highest antibody concentrations. Results We observed different persistent fractions for NAb neutralization of pseudovirus derived from two Tier-2 isolates of HIV-1, BG505 (Clade A) and B41 (Clade B): it was pronounced for B41 but not BG505 neutralization by NAb PGT151, directed to the interface between the outer and transmembrane subunits of Env, but negligible for either virus by NAb PGT145 to an apical epitope. Autologous neutralization by poly- and monoclonal NAbs from rabbits immunized with soluble native-like B41 trimer also left substantial persistent fractions. These NAbs largely target a cluster of epitopes in a hole in the dense glycan shield of Env around residue 289. We partially depleted B41-virion populations by incubating them with PGT145- or PGT151-conjugated beads. Each depletion reduced the sensitivity to the depleting NAb and enhanced it to the other. Autologous neutralization by the rabbit NAbs was reduced for PGT145-depleted and enhanced for PGT151-depleted B41 pseudovirus. Those changes in sensitivity encompassed both potency and the persistent fraction. We then compared soluble native-like BG505 and B41 Env trimers affinity-purified by one of three NAbs: 2G12, PGT145, or PGT151. Surface plasmon resonance showed differences among the fractions in antigenicity, including kinetics and stoichiometry, congruently with the differential neutralization. The large persistent fraction after PGT151 neutralization of B41 was attributable to low stoichiometry, which we explained structurally by the conformational plasticity of B41 Env. Conclusion Distinct antigenic forms even of clonal HIV-1 Env, detectable among soluble native-like trimer molecules, are distributed over virions and may profoundly mold neutralization of certain isolates by certain NAbs. Affinity purifications with some antibodies may yield immunogens that preferentially expose epitopes for broadly active NAbs, while shielding less cross-reactive ones. NAbs reactive with multiple conformers will together reduce the persistent fraction after passive and active immunization.

Measurements of SARS-CoV-2 antibody dissociation rate constant by chaotrope-free biolayer interferometry in serum of COVID-19 convalescent patients.

Kinetics measurements of antigen-antibody binding interactions are critical to understanding the functional efficiency of SARS-CoV-2 antibodies. Previously reported chaotrope-based avidity assays that rely on artificial disruption of binding do not reflect the natural binding kinetics. This study developed a chaotrope- and label-free biolayer interferometry (BLI) assay for the real-time monitoring of receptor binding domain (RBD) binding kinetics with SARS-CoV-2 spike protein in convalescent COVID-19 patients. An improved conjugation biosensor probe coated with streptavidin-polysaccharide (SA-PS) led to a six-fold increase of signal intensities and two-fold reduction of non-specific binding (NSB) compared to streptavidin only probe. Furthermore, by utilizing a separate reference probe and biotin-human serum albumin (B-HSA) blocking process to subtracted NSB signal in serum, this BLI biosensor can measure a wide range of the dissociation rate constant (koff), which can be measured without knowledge of the specific antibody concentrations. The clinical utility of this improved BLI kinetics assay was demonstrated by analyzing the koff values in sera of 24 pediatric (≤18 years old) and 63 adult (>18 years old) COVID-19 convalescent patients. Lower koff values for SARS-CoV-2 serum antibodies binding to RBD were measured in samples from children. This rapid, easy to operate and chaotrope-free BLI assay is suitable for clinical use and can be readily adapted to characterize SARS-CoV-2 antibodies developed by COVID-19 patients and vaccines.

Reappraising the Value of HIV-1 Vaccine Correlates of Protection Analyses.

With the much-debated exception of the modestly reduced acquisition reported for the RV144 efficacy trial, HIV-1 vaccines have not protected humans against infection, and a vaccine of similar design to that tested in RV144 was not protective in a later trial, HVTN 702. Similar vaccine regimens have also not consistently protected nonhuman primates (NHPs) against viral acquisition. Conversely, experimental vaccines of different designs have protected macaques from viral challenges but then failed to protect humans, while many other HIV-1 vaccine candidates have not protected NHPs. While efficacy varies more in NHPs than humans, vaccines have failed to protect in the most stringent NHP model. Intense investigations have aimed to identify correlates of protection (CoPs), even in the absence of net protection. Unvaccinated animals and humans vary vastly in their susceptibility to infection and in their innate and adaptive responses to the vaccines; hence, merely statistical associations with factors that do not protect are easily found. Systems biological analyses, including artificial intelligence, have identified numerous candidate CoPs but with no clear consistency within or between species. Proposed CoPs sometimes have only tenuous mechanistic connections to immune protection. In contrast, neutralizing antibodies (NAbs) are a central mechanistic CoP for vaccines that succeed against other viruses, including SARS-CoV-2. No HIV-1 vaccine candidate has yet elicited potent and broadly active NAbs in NHPs or humans, but narrow-specificity NAbs against the HIV-1 isolate corresponding to the immunogen do protect against infection by the autologous virus. Here, we analyze why so many HIV-1 vaccines have failed, summarize the outcomes of vaccination in NHPs and humans, and discuss the value and pitfalls of hunting for CoPs other than NAbs. We contrast the failure to find a consistent CoP for HIV-1 vaccines with the identification of NAbs as the principal CoP for SARS-CoV-2.

The Glycan Hole Area of HIV-1 Envelope Trimers Contributes Prominently to the Induction of Autologous Neutralization.

The human immunodeficiency virus type 1 (HIV-1) trimeric envelope glycoprotein (Env) is heavily glycosylated, creating a dense glycan shield that protects the underlying peptidic surface from antibody recognition. The absence of conserved glycans, due to missing potential N-linked glycosylation sites (PNGS), can result in strain-specific, autologous neutralizing antibody (NAb) responses. Here, we sought to gain a deeper understanding of the autologous neutralization by introducing holes in the otherwise dense glycan shields of the AMC011 and AMC016 SOSIP trimers. Specifically, when we knocked out the N130 and N289 glycans, which are absent from the well-characterized B41 SOSIP trimer, we observed stronger autologous NAb responses. We also analyzed the highly variable NAb responses induced in rabbits by diverse SOSIP trimers from subtypes A, B, and C. Statistical analysis, using linear regression, revealed that the cumulative area exposed on a trimer by glycan holes correlates with the magnitude of the autologous NAb response. IMPORTANCE Forty years after the first description of HIV-1, the search for a protective vaccine is still ongoing. The sole target for antibodies that can neutralize the virus are the trimeric envelope glycoproteins (Envs) located on the viral surface. The glycoprotein surface is covered with glycans that shield off the underlying protein components from recognition by the immune system. However, the Env trimers of some viral strains have holes in the glycan shield. Immunized animals developed antibodies against such glycan holes. These antibodies are generally strain specific. Here, we sought to gain a deeper understanding of what drives these specific immune responses. First, we show that strain-specific neutralizing antibody responses can be increased by creating artificial holes in the glycan shield. Second, when studying a diverse set of Env trimers with different characteristics, we found that the surface area of the glycan holes contributes prominently to the induction of strain-specific neutralizing antibodies.

Antibodies from Rabbits Immunized with HIV-1 Clade B SOSIP Trimers Can Neutralize Multiple Clade B Viruses by Destabilizing the Envelope Glycoprotein.

The high viral diversity of HIV-1 is a formidable hurdle for the development of an HIV-1 vaccine. Elicitation of broadly neutralizing antibodies (bNAbs) would offer a solution, but so far immunization strategies have failed to efficiently elicit bNAbs. To overcome these obstacles, it is important to understand the immune responses elicited by current HIV-1 envelope glycoprotein (Env) immunogens. To gain more insight, we characterized monoclonal antibodies (MAbs) isolated from rabbits immunized with Env SOSIP trimers based on the clade B isolate AMC008. Four rabbits that were immunized three times with AMC008 trimer developed robust autologous and sporadic low-titer heterologous neutralizing responses. Seventeen AMC008 trimer-reactive MAbs were isolated using antigen-specific single B-cell sorting. Four of these MAbs neutralized the autologous AMC008 virus and several other clade B viruses. When visualized by electron microscopy, the complex of the neutralizing MAbs with the AMC008 trimer showed binding to the gp41 subunit with unusual approach angles, and we observed that their neutralization ability depended on their capacity to induce Env trimer dissociation. Thus, AMC008 SOSIP trimer immunization induced clade B-neutralizing MAbs with unusual approach angles with neutralizing effects that involve trimer destabilization. Optimizing these responses might provide an avenue to the induction of trimer-dissociating bNAbs. IMPORTANCE Roughly 32 million people have died as a consequence of HIV-1 infection since the start of the epidemic, and 1.7 million people still get infected with HIV-1 annually. Therefore, a vaccine to prevent HIV-1 infection is urgently needed. Current HIV-1 immunogens are not able to elicit the broad immune responses needed to provide protection against the large variation of HIV-1 strains circulating globally. A better understanding of the humoral immune responses elicited by immunization with state-of-the-art HIV-1 immunogens should facilitate the design of improved HIV-1 vaccine candidates. We identified antibodies with the ability to neutralize multiple HIV-1 viruses by destabilization of the envelope glycoprotein. Their weak but consistent cross-neutralization ability indicates the potential of this epitope to elicit broad responses. The trimer-destabilizing effect of the neutralizing MAbs, combined with detailed characterization of the neutralization epitope, can be used to shape the next generation of HIV-1 immunogens to elicit improved humoral responses after vaccination.

TOP-Plus Is a Versatile Biosensor Platform for Monitoring SARS-CoV-2 Antibody Durability.

BACKGROUND: Low initial severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody titers dropping to undetectable levels within months after infection have raised concerns about long-term immunity. Both the antibody levels and the avidity of the antibody-antigen interaction should be examined to understand the quality of the antibody response. METHODS: A testing-on-a-probe "plus" panel (TOP-Plus) was developed to include a newly developed avidity assay built into the previously described SARS-CoV-2 TOP assays that measured total antibody (TAb), surrogate neutralizing antibody (SNAb), IgM, and IgG on a versatile biosensor platform. TAb and SNAb levels were compared with avidity in previously infected individuals at 1.3 and 6.2 months after infection in paired samples from 80 patients with coronavirus disease 2019 (COVID-19). Sera from individuals vaccinated for SARS-CoV-2 were also evaluated for antibody avidity. RESULTS: The newly designed avidity assay in this TOP panel correlated well with a reference Bio-Layer Interferometry avidity assay (r = 0.88). The imprecision of the TOP avidity assay was <10%. Although TAb and neutralization activity (by SNAb) decreased between 1.3 and 6.2 months after infection, the antibody avidity increased significantly (P < 0.0001). Antibody avidity in 10 SARS-CoV-2 vaccinated individuals (median: 28 days after vaccination) was comparable to the measured antibody avidity in infected individuals (median: 26 days after infection). CONCLUSIONS: This highly precise and versatile TOP-Plus panel with the ability to measure SARS-CoV-2 TAb, SNAb, IgG, and IgM antibody levels and avidity of individual sera on one sensor can become a valuable asset in monitoring not only patients infected with SARS-CoV-2 but also the status of individuals' COVID-19 vaccination response.

Enhancing glycan occupancy of soluble HIV-1 envelope trimers to mimic the native viral spike.

Artificial glycan holes on recombinant Env-based vaccines occur when a potential N-linked glycosylation site (PNGS) is under-occupied, but not on their viral counterparts. Native-like SOSIP trimers, including clinical candidates, contain such holes in the glycan shield that induce strain-specific neutralizing antibodies (NAbs) or non-NAbs. To eliminate glycan holes and mimic the glycosylation of native BG505 Env, we replace all 12 NxS sequons on BG505 SOSIP with NxT. All PNGS, except N133 and N160, are nearly fully occupied. Occupancy of the N133 site is increased by changing N133 to NxS, whereas occupancy of the N160 site is restored by reverting the nearby N156 sequon to NxS. Hence, PNGS in close proximity, such as in the N133-N137 and N156-N160 pairs, affect each other's occupancy. We further apply this approach to improve the occupancy of several Env strains. Increasing glycan occupancy should reduce off-target immune responses to vaccine antigens.

Association of Age With SARS-CoV-2 Antibody Response.

Importance: Accumulating evidence suggests that children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more likely to manifest mild symptoms and are at a lower risk of developing severe respiratory disease compared with adults. It remains unknown how the immune response in children differs from that of adolescents and adults. Objective: To investigate the association of age with the quantity and quality of SARS-CoV-2 antibody responses. Design, Setting, and Participants: This cross-sectional study used 31 426 SARS-CoV-2 antibody test results from pediatric and adult patients. Data were collected from a New York City hospital from April 9 to August 31, 2020. The semiquantitative immunoglobin (Ig) G levels were compared between 85 pediatric and 3648 adult patients. Further analysis of SARS-CoV-2 antibody profiles was performed on sera from 126 patients aged 1 to 24 years. Main Outcomes and Measures: SARS-CoV-2 antibody positivity rates and IgG levels were evaluated in patients from a wide range of age groups (1-102 years). SARS-CoV-2 IgG level, total antibody (TAb) level, surrogate neutralizing antibody (SNAb) activity, and antibody binding avidity were compared between children (aged 1-10 years), adolescents (aged 11-18 years), and young adults (aged 19-24 years). Results: Among 31 426 antibody test results (19 797 [63.0%] female patients), with 1194 pediatric patients (mean [SD] age, 11.0 [5.3] years) and 30 232 adult patients (mean [SD] age, 49.2 [17.1] years), the seroprevalence in the pediatric (197 [16.5%; 95% CI, 14.4%-18.7%]) and adult (5630 [18.6%; 95% CI, 18.2%-19.1%]) patient populations was similar. The SARS-CoV-2 IgG level showed a negative correlation with age in the pediatric population (r = -0.45, P < .001) and a moderate but positive correlation with age in adults (r = 0.24, P < .001). Patients aged 19 to 30 years exhibited the lowest IgG levels (eg, aged 25-30 years vs 1-10 years: 99 [44-180] relative fluorescence units [RFU] vs 443 [188-851] RFU). In the subset cohort aged 1 to 24 years, IgG, TAb, SNAb and avidity were negatively correlated with age (eg, IgG: r = -0.51; P < .001). Children exhibited higher median (IQR) IgG levels, TAb levels, and SNAb activity compared with adolescents (eg, IgG levels: 473 [233-656] RFU vs 191 [82-349] RFU; P < .001) and young adults (eg, IgG levels: 473 [233-656] RFU vs 85 [38-150] RFU; P < .001). Adolescents also exhibited higher median (IQR) TAb levels, IgG levels, and SNAb activity than young adults (eg, TAb levels: 961 [290-2074] RFU vs 370 [125-697]; P = .006). In addition, children had higher antibody binding avidity compared with young adults, but the difference was not significant. Conclusions and Relevance: The results of this study suggest that SARS-CoV-2 viral specific antibody response profiles are distinct in different age groups. Age-targeted strategies for disease screening and management as well as vaccine development may be warranted.

Antibody responses to SARS-CoV-2 mRNA vaccines are detectable in saliva.

Vaccines are critical for curtailing the COVID-19 pandemic (1, 2). In the USA, two highly protective mRNA vaccines are available: BNT162b2 from Pfizer/BioNTech and mRNA-1273 from Moderna (3, 4). These vaccines induce antibodies to the SARS-CoV-2 S-protein, including neutralizing antibodies (NAbs) predominantly directed against the Receptor Binding Domain (RBD) (1-4). Serum NAbs are induced at modest levels within ~1 week of the first dose, but their titers are strongly boosted by a second dose at 3 (BNT162b2) or 4 weeks (mRNA-1273) (3, 4). SARS-CoV-2 is most commonly transmitted nasally or orally and infects cells in the mucosae of the respiratory and to some extent also the gastrointestinal tract (5). Although serum NAbs may be a correlate of protection against COVID-19, mucosal antibodies might directly prevent or limit virus acquisition by the nasal, oral and conjunctival routes (5). Whether the mRNA vaccines induce mucosal immunity has not been studied. Here, we report that antibodies to the S-protein and its RBD are present in saliva samples from mRNA-vaccinated healthcare workers (HCW). Within 1-2 weeks after their second dose, 37/37 and 8/8 recipients of the Pfizer and Moderna vaccines, respectively, had S-protein IgG antibodies in their saliva, while IgA was detected in a substantial proportion. These observations may be relevant to vaccine-mediated protection from SARS-CoV-2 infection and disease.

Postconvalescent SARS-CoV-2 IgG and Neutralizing Antibodies are Elevated in Individuals with Poor Metabolic Health.

PURPOSE: Comorbidities making up metabolic syndrome (MetS), such as obesity, type 2 diabetes, and chronic cardiovascular disease can lead to increased risk of coronavirus disease-2019 (COVID-19) with a higher morbidity and mortality. SARS-CoV-2 antibodies are higher in severely or critically ill COVID-19 patients, but studies have not focused on levels in convalescent patients with MetS, which this study aimed to assess. METHODS: This retrospective study focused on adult convalescent outpatients with SARS-CoV-2 positive serology during the COVID-19 pandemic at NewYork Presbyterian/Weill Cornell. Data collected for descriptive and correlative analysis included SARS-COV-2 immunoglobin G (IgG) levels and history of MetS comorbidities from April 17, 2020 to May 20, 2020. Additional data, including SARS-CoV-2 IgG levels, body mass index (BMI), hemoglobin A1c (HbA1c) and lipid levels were collected and analyzed for a second cohort from May 21, 2020 to June 21, 2020. SARS-CoV-2 neutralizing antibodies were measured in a subset of the study cohort. RESULTS: SARS-CoV-2 IgG levels were significantly higher in convalescent individuals with MetS comorbidities. When adjusted for age, sex, race, and time duration from symptom onset to testing, increased SARS-CoV-2 IgG levels remained significantly associated with obesity (P < 0.0001). SARS-CoV-2 IgG levels were significantly higher in patients with HbA1c ≥6.5% compared to those with HbA1c <5.7% (P = 0.0197) and remained significant on multivariable analysis (P = 0.0104). A positive correlation was noted between BMI and antibody levels [95% confidence interval: 0.37 (0.20-0.52) P < 0.0001]. Neutralizing antibody titers were higher in COVID-19 individuals with BMI ≥ 30 (P = 0.0055). CONCLUSION: Postconvalescent SARS-CoV-2 IgG and neutralizing antibodies are elevated in obese patients, and a positive correlation exists between BMI and antibody levels.

Immunogenicity of clinically relevant SARS-CoV-2 vaccines in nonhuman primates and humans.

Multiple preventive vaccines are being developed to counter the coronavirus disease 2019 pandemic. The leading candidates have now been evaluated in nonhuman primates (NHPs) and human phase 1 and/or phase 2 clinical trials. Several vaccines have already advanced into phase 3 efficacy trials, while others will do so before the end of 2020. Here, we summarize what is known of the antibody and T cell immunogenicity of these vaccines in NHPs and humans. To the extent possible, we compare how the vaccines have performed, taking into account the use of different assays to assess immunogenicity and inconsistencies in how the resulting data are presented. We also review the outcome of challenge experiments with severe acute respiratory syndrome coronavirus 2 in immunized macaques, while noting variations in the protocols used, including but not limited to the virus challenge doses. Press releases on the outcomes of vaccine efficacy trials are also summarized.

TOP-Plus is a Versatile Biosensor Platform for Monitoring SARS-CoV-2 Antibody Durability.

BACKGROUND: There is a concern that low initial SARS-CoV-2 antibody titers in individuals may drop to undetectable levels within months after infection. Although this may raise concerns over long term immunity, both the antibody levels and avidity of the antibody-antigen interaction should be examined to understand the quality of the antibody response. METHODS: A testing-on-a-probe-plus panel (TOP-Plus) was developed, which included a newly developed avidity assay built into the previously described SARS-CoV-2 TOP assays that measured total antibody (TAb), surrogate neutralizing antibody (SNAb), IgM and IgG on a versatile biosensor platform. TAb and SNAb levels were compared with avidity in previously infected individuals at 1.3 and 6.2 months post-infection in paired samples from 80 COVID-19 patients. RESULTS: The newly designed avidity assay in this TOP panel correlated well with a reference Bio-Layer Interferometry avidity assay (R=0.88). The imprecision of the TOP avidity assay was less than 9%. Although TAb and neutralization activity (by SNAb) decreased between 1.3 and 6.2 months post infection, the antibody avidity increased significantly (P < 0.0001).

High-resolution mapping of the neutralizing and binding specificities of polyclonal sera post-HIV Env trimer vaccination.

Mapping polyclonal serum responses is critical to rational vaccine design. However, most high-resolution mapping approaches involve isolating and characterizing individual antibodies, which incompletely defines the polyclonal response. Here we use two complementary approaches to directly map the specificities of the neutralizing and binding antibodies of polyclonal anti-HIV-1 sera from rabbits immunized with BG505 Env SOSIP trimers. We used mutational antigenic profiling to determine how all mutations in Env affected viral neutralization and electron microscopy polyclonal epitope mapping (EMPEM) to directly visualize serum Fabs bound to Env trimers. The dominant neutralizing specificities were generally only a subset of the more diverse binding specificities. Additional differences between binding and neutralization reflected antigenicity differences between virus and soluble Env trimer. Furthermore, we refined residue-level epitope specificity directly from sera, revealing subtle differences across sera. Together, mutational antigenic profiling and EMPEM yield a holistic view of the binding and neutralizing specificity of polyclonal sera.

Vaccines work by stimulating the immune system to produce proteins called antibodies. These antibodies bind to the virus targeted by the vaccine and block the virus from infecting cells. It has been difficult to develop a vaccine for HIV because frequent mutations allow it to evade antibodies. Understanding exactly how these proteins bind to HIV and how various mutations enable the virus to escape them is crucial to designing a successful HIV vaccine. Over the last decade, scientists have developed new techniques for studying individual antibodies and how they bind to viruses. Now, they are using these insights to design vaccines. Most vaccines result in the production of many antibodies that bind to different parts of the virus, making it harder for a virus to escape. But studying many antibodies with different targets on the virus simultaneously remains challenging. By combining two-cutting edge approaches, Dingens et al. catalogued the many antibodies that rabbits produce in response to an experimental vaccine for HIV. In the experiments, they mapped how two types of rabbit antibodies target the virus: those that could bind to the virus, and those that could both bind and neutralize the virus (i.e., block it from infecting cells). The experiments showed that small differences between the HIV virus and the vaccine explained why some rabbit antibodies created in response to the vaccine could bind but not neutralize the virus. Moreover, the ability to stop HIV from infecting the cells appeared to be reserved to antibodies that could bind to several different locations at the virus. Dingens et al. further documented all the virus mutations that would allow it to evade neutralizing antibodies. The techniques used in the experiments may help scientists identify the best sites on the HIV virus to target with vaccines and to better understand the binding and neutralizing activity of antibodies. The results of the experiments may also help to redesign the experimental HIV vaccine ­ which is currently being tested in humans ­ to be even more effective.

Testing-on-a-probe biosensors reveal association of early SARS-CoV-2 total antibodies and surrogate neutralizing antibodies with mortality in COVID-19 patients.

The association of mortality with the early humoral response to SARS-CoV-2 infection within the first few days after onset of symptoms (DAOS) has not been thoroughly investigated partly due to a lack of sufficiently sensitive antibody testing methods. Here we report two sensitive and automated testing-on-a-probe (TOP) biosensor assays for SARS-CoV-2 viral specific total antibodies (TAb) and surrogate neutralizing antibodies (SNAb), which are suitable for clinical use. The TOP assays employ an RBD-coated quartz probe using a Cy5-Streptavidin-polysacharide conjugate to improve sensitivity and minimize interference. Disposable cartridges containing pre-dispensed reagents require no liquid manipulation or fluidics during testing. The TOP-TAb assay exhibited higher sensitivity in the 0-7 DAOS window than a widely used FDA-EUA assay. The rapid and automated TOP-SNAb correlated well with two well-established SARS-CoV-2 virus neutralization tests. The clinical utility of the TOP assays was demonstrated by evaluating early antibody responses in 120 SARS-CoV-2 RT-PCR positive adult hospitalized patients. Higher TAb and SNAb positivity rates and more robust antibody responses at patient's initial hospital presentation were seen in inpatients who survived COVID-19 than those who died in the hospital. Survival analysis using the Cox Proportional Hazards Model showed that patients who had negative TAb and/or SNAb at initial hospital presentation were at a higher risk of in-hospital mortality. Furthermore, TAb and SNAb levels at presentation were inversely associated with SARS-CoV-2 viral load based on concurrent RT-PCR testing. Overall, the sensitive and automated TAb and SNAb assays allow the detection of early SARS-CoV-2 antibodies which associate with mortality.

Testing-on-a-probe biosensors reveal association of early SARS-CoV-2 total antibodies and surrogate neutralizing antibodies with mortality in COVID-19 patients.

The association of mortality with early humoral response to SARS-CoV-2 infection within the first few days after onset of symptoms (DAOS) has not been thoroughly investigated partly due to a lack of sufficiently sensitive antibody testing methods. Here we report two sensitive and automated testing-on-a-probe (TOP) biosensor assays for SARS-CoV-2 viral specific total antibodies (TAb) and surrogate neutralizing antibodies (SNAb), which are suitable for clinical use. The TOP assays employ an RBD-coated quartz probe using a Cy5-Streptavidin-polysacharide conjugate to improved sensitivity and minimize interference. Disposable cartridge containing pre-dispensed reagents requires no liquid manipulation or fluidics during testing. The TOP-TAb assay exhibited higher sensitivity in the 0-7 DAOS window than a widely used FDA-EUA assay. The rapid (18 min) and automated TOP-SNAb correlated well with two well-established SARS-CoV-2 virus neutralization tests. The clinical utility of the TOP assays was demonstrated by evaluating early antibody responses in 120 SARS-CoV-2 RT-PCR positive adult hospitalized patients. Higher baseline TAb and SNAb positivity rates and more robust antibody responses were seen in patients who survived COVID-19 than those who died in the hospital. Survival analysis using the Cox Proportional Hazards Model showed that patients who were TAb and SNAb negative at initial hospital presentation were at a higher risk of in-hospital mortality. Furthermore, TAb and SNAb levels at presentation were inversely associated with SARS-CoV-2 viral load based on concurrent RT-PCR testing. Overall, the sensitive and automated TAb and SNAb assays allow detection of early SARS-CoV-2 antibodies which associate with mortality.

Antibodies to SARS-CoV-2 and their potential for therapeutic passive immunization.

We review aspects of the antibody response to SARS-CoV-2, the causative agent of the COVID-19 pandemic. The topics we cover are relevant to immunotherapy with plasma from recovered patients, monoclonal antibodies against the viral S-protein, and soluble forms of the receptor for the virus, angiotensin converting enzyme 2. The development of vaccines against SARS-CoV-2, an essential public health tool, will also be informed by an understanding of the antibody response in infected patients. Although virus-neutralizing antibodies are likely to protect, antibodies could potentially trigger immunopathogenic events in SARS-CoV-2-infected patients or enhance infection. An awareness of these possibilities may benefit clinicians and the developers of antibody-based therapies and vaccines.