Three doses of BNT162b2 vaccine confer neutralising antibody capacity against the SARS-CoV-2 Omicron variant.

We report the levels of neutralising antibodies against Wuhan, Delta and Omicron variants in unimmunized infected (group 1), immunised and boosted (group 2) and infected immunised and boosted (group 3) adult individuals. Our observations support the rapid administration of a booster vaccine dose to prevent infection and disease caused by Omicron.

Predictors of neutralizing antibody response to BNT162b2 vaccination in allogeneic hematopoietic stem cell transplant recipients.

BACKGROUND: Factors affecting response to SARS-CoV-2 mRNA vaccine in allogeneic hematopoietic stem cell transplantation (allo-HCT) recipients remain to be elucidated. METHODS: Forty allo-HCT recipients were included in a study of immunization with BNT162b2 mRNA vaccine at days 0 and 21. Binding antibodies (Ab) to SARS-CoV-2 receptor binding domain (RBD) were assessed at days 0, 21, 28, and 49 while neutralizing Ab against SARS-CoV-2 wild type (NT50) were assessed at days 0 and 49. Results observed in allo-HCT patients were compared to those obtained in 40 healthy adults naive of SARS-CoV-2 infection. Flow cytometry analysis of peripheral blood cells was performed before vaccination to identify potential predictors of Ab responses. RESULTS: Three patients had detectable anti-RBD Ab before vaccination. Among the 37 SARS-CoV-2 naive patients, 20 (54%) and 32 (86%) patients had detectable anti-RBD Ab 21 days and 49 days postvaccination. Comparing anti-RBD Ab levels in allo-HCT recipients and healthy adults, we observed significantly lower anti-RBD Ab levels in allo-HCT recipients at days 21, 28 and 49. Further, 49% of allo-HCT patients versus 88% of healthy adults had detectable NT50 Ab at day 49 while allo-HCT recipients had significantly lower NT50 Ab titers than healthy adults (P = 0.0004). Ongoing moderate/severe chronic GVHD (P < 0.01) as well as rituximab administration in the year prior to vaccination (P < 0.05) correlated with low anti-RBD and NT50 Ab titers at 49 days after the first vaccination in multivariate analyses. Compared to healthy adults, allo-HCT patients without chronic GVHD or rituximab therapy had comparable anti-RBD Ab levels and NT50 Ab titers at day 49. Flow cytometry analyses before vaccination indicated that Ab responses in allo-HCT patients were strongly correlated with the number of memory B cells and of naive CD4+ T cells (r > 0.5, P < 0.01) and more weakly with the number of follicular helper T cells (r = 0.4, P = 0.01). CONCLUSIONS: Chronic GVHD and rituximab administration in allo-HCT recipients are associated with reduced Ab responses to BNT162b2 vaccination. Immunological markers could help identify allo-HCT patients at risk of poor Ab response to mRNA vaccination. TRIAL REGISTRATION: The study was registered at clinicaltrialsregister.eu on 11 March 2021 (EudractCT # 2021-000673-83).

International network for comparison of HIV neutralization assays: the NeutNet report II.

BACKGROUND: Neutralizing antibodies provide markers for vaccine-induced protective immunity in many viral infections. By analogy, HIV-1 neutralizing antibodies induced by immunization may well predict vaccine effectiveness. Assessment of neutralizing antibodies is therefore of primary importance, but is hampered by the fact that we do not know which assay(s) can provide measures of protective immunity. An international collaboration (NeutNet) involving 18 different laboratories previously compared different assays using monoclonal antibodies (mAbs) and soluble CD4 (Phase I study). METHODS: In the present study (Phase II), polyclonal reagents were evaluated by 13 laboratories. Each laboratory evaluated nine plasmas against an 8 virus panel representing different genetic subtypes and phenotypes. TriMab, a mixture of three mAbs, was used as a positive control allowing comparison of the results with Phase I in a total of nine different assays. The assays used either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (Virus Infectivity Assays, VIA), or Env (gp160)-pseudotyped viruses (pseudoviruses, PSV) produced in HEK293T cells from molecular clones or from uncloned virus. Target cells included PBMC and genetically engineered cell lines in either single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs including extra- or intra-cellular p24 antigen detection, luciferase, beta-galactosidase or green fluorescent protein (GFP) reporter gene expression. FINDINGS: Using TriMab, results of Phase I and Phase II were generally in agreement for six of the eight viruses tested and confirmed that the PSV assay is more sensitive than PBMC (p = 0.014). Comparisons with the polyclonal reagents showed that sensitivities were dependent on both virus and plasma. CONCLUSIONS: Here we further demonstrate clear differences in assay sensitivities that were dependent on both the neutralizing reagent and the virus. Consistent with the Phase I study, we recommend parallel use of PSV and VIA for vaccine evaluation.

Characterization of neutralizing profiles in HIV-1 infected patients from whom the HJ16, HGN194 and HK20 mAbs were obtained.

Several new human monoclonal antibodies (mAbs) with a neutralizing potential across different subtypes have recently been described. Three mAbs, HJ16, HGN194 and HK20, were obtained from patients within the HIV-1 cohort of the Institute of Tropical Medicine (ITM). Our aim was to generate immunization antibodies equivalent to those seen in plasma. Here, we describe the selection and characterization of patient plasma and their mAbs, using a range of neutralization assays, including several peripheral blood mononuclear cell (PBMC) based assays and replicating primary viruses as well as cell line based assays and pseudoviruses (PV). The principal criterion for selection of patient plasma was the activity in an 'extended incubation phase' PBMC assay. Neutralizing Abs, derived from their memory B cells, were then selected by ELISA with envelope proteins as solid phase. MAbs were subsequently tested in a high-throughput HOS-PV assay to assess functional neutralization. The present study indicates that the strong profiles in the patients' plasma were not solely due to antibodies represented by the newly isolated mAbs. Although results from the various assays were divergent, they by and large indicate that neutralizing Abs to other epitopes of the HIV-1 envelope are present in the plasma and synergy between Abs may be important. Thus, the spectrum of the obtained mAbs does not cover the range of cross-reactivity seen in plasma in these carefully selected patients irrespective of which neutralization assay is used. Nevertheless, these mAbs are relevant for immunogen discovery because they bind to the recombinant glycoproteins to which the immune response needs to be targeted in vivo. Our observations illustrate the remaining challenges required for successful immunogen design and development.

Unravelling the antigenic landscape of the HIV-1 subtype A envelope of an individual with broad cross-neutralizing antibodies using phage display peptide libraries.

Broad cross-neutralizing antibodies from persons infected with HIV-1 target a variety of epitopes. Identification of these HIV-1 epitopes may result in an optimal panel of antigenic peptides to be included in a prophylactic vaccine. Phage display peptide libraries were used to unravel the antigenic landscape of an individual (ITM1) infected with HIV-1 subtype A with broad cross-neutralizing antibodies. A stringent selection strategy resulted in the identification of 60 unique HIV-1 peptide phage, which were subjected to sequence analysis and mapped onto the ITM1 envelope sequences. Four groups of peptide phages were found: the first group (n=11) were similar with the tip of the V3 loop (KxxHxGPxxxF); the second group (n=11) represented the gp41 principal immunodominant domain (CxGxLxCTxNxP); the third group (n=16) could be localized in the V2 loop (KxxxHxxxY); and the fourth group (n=22) mimicked a conformational epitope (Hxx(S)/(T)NxK). All but the V2-binding antibodies were conserved over the 11 years of follow-up. A neutralization inhibition assay revealed the contribution of the V3 antibodies to the neutralizing capacity of the ITM1 plasma. Overall, the ITM1 immunogenic landscape was mapped and a part of the origin of this broad cross-neutralizing activity was demonstrated.

Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals.

BACKGROUND: The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine. METHODS AND FINDINGS: We immortalized IgG(+) memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env) in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16) specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194) bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20) with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity. CONCLUSIONS: This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.

Evolution of antibody landscape and viral envelope escape in an HIV-1 CRF02_AG infected patient with 4E10-like antibodies.

BACKGROUND: A minority of HIV-1 infected individuals develop broad cross-neutralizing (BCN) plasma antibodies that are capable of neutralizing a spectrum of virus variants belonging to different HIV-1 clades. The aim of this study was to identify the targeted epitopes of an individual with BCN plasma antibodies, referred to as ITM4, using peptide phage display. This study also aimed to use the selected mimotopes as tools to unravel the evolution of the antibody landscape and the viral envelope escape which may provide us with new insights for vaccine design. RESULTS: This study led us to identify ITM4 plasma antibodies directed to the 4E10 epitope located in the gp41 membrane-proximal external region (MPER). Analysis of antibody specificities revealed unusual immunogenic properties of the ITM4 viral envelope, as not only the V3 loop and the gp41 MPER but also the C1 and lentivirus lytic peptide 2 (LLP2) region seem to be targets of the immune system. The 4E10-like antibodies are consistently elicited during the 6-year follow up period. HIV-1 ITM4 pseudoviruses showed an increasing resistance over time to MPER monoclonal antibodies 4E10 and 2F5, although no changes are found in the critical positions of the epitope. Neutralization of COT6.15 (subtype C; 4E10-sensitive) pseudoviruses with alanine substitutions in the MPER region indicated an overlapping specificity of the 4E10 monoclonal antibody and the ITM4 follow up plasma. Moreover the 4E10-like antibodies of ITM4 contribute to the BCN capacity of the plasma. CONCLUSIONS: Using ITM4 BCN plasma and peptide phage display technology, we have identified a patient with 4E10-like BCN antibodies. Our results indicate that the elicited 4E10-like antibodies play a role in virus neutralization. The viral RNA was isolated at different time points and the ITM4 envelope sequence analysis of both early (4E10-sensitive) and late (4E10-resistant) viruses suggest that other regions in the envelope, outside the MPER region, contribute to the accessibility and sensitivity of the 4E10 epitope. Including ITM4 specific HIV-1 Env properties in vaccine strategies may be a promising approach.

Neutralization kinetics of sensitive and resistant subtype B primary human immunodeficiency virus type 1 isolates.

The aim of the study was to determine if sensitive and resistant human immunodeficiency virus type 1 (HIV-1) subtype B primary isolates have different neutralization kinetics. Neutralization assays were undertaken where either the time allowed for virus to react with antibodies or the subsequent period of this mixture's exposure to target cells were varied. The relative neutralization sensitivity/resistance is a reproducible property of the isolates. In a minority of combinations, the titre falls exponentially for as long as the free virions are exposed to antibody. In the remainder, neutralization kinetics shows deviations which may be attributed to events occurring after the virus-antibody mixture is added to the target cells: significant neutralization with minimal exposure of the free virions to antibody; a plot where reduction in virus titre is parallel to the duration of the incubation phase of the assay. Neutralization rate constants are similar for primary HIV-1 SF33, HIV-1 SF162, and HIV-1 89.6, reaching 5 x 10(5)-1 x 10(6)/M sec for the monoclonal antibody IgG1 b12. However, although increased antibody levels produced greater reductions in virus titre the rate of neutralization was not proportional to the antibody concentration. Neutralization of either the free virion or cell-associated virus does not correlate with the resistance/sensitivity of primary subtype B isolates. The target cells play an active role, so that in designing neutralization assays with primary isolates of HIV-1, events following the virus-antibody complex binding to the cell surface have to be taken into consideration.

Neutralization of primary HIV-1 SF13 can be detected in extended incubation phase assays with sera from monkeys immunized with recombinant HIV-1 SF2 gp120.

Phase III efficacy trials of recombinant human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins were postponed. In Phase I and II trials these candidate vaccines had failed to induce neutralizing antibodies against virus which had been isolated by co-culture with human peripheral blood mononuclear cells (PBMC). The aim of the present study was to determine assay conditions for detecting neutralization of primary HIV-1 isolates with sera from immunized individuals. We show that in two immunogenicity trials in rhesus macaques, recombinant HIV-1 SF2 gp120 induced antibodies which neutralized the primary HIV-1 SF13 isolate. Statistically significant in vitro neutralization required assays in which the incubation phase was extended. Sterile immunity was only seen with the highest level of neutralization, induced by a recombinant prime, peptide boost strategy. We recommend that neutralization assays with extended incubation phases should be used to monitor Phase III efficacy trials.

The first generation of candidate HIV-1 vaccines can induce antibodies able to neutralize primary isolates in assays with extended incubation phases.

Quantification of human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies is considered to be an important parameter in evaluating candidate vaccines. Most previous studies have failed to detect vaccine-induced antibodies against primary isolates, which are more resistant to antibody mediated neutralization compared with laboratory isolates, in neutralization assays. In this study, sera from a prime boost vaccination strategy of a phase I clinical trial were tested against six clade B primary HIV-1 isolates and single isolates of clades C and F. These sera produced statistically significant neutralization against primary isolates MN, SF13, SF162 and Han 2 but not the most resistant subtype B isolates (92US077 and 93US143) nor the subtype C and F isolates. These data suggest that the sera from vaccinated volunteers have subtype-specific neutralizing antibodies against primary HIV-1 isolates. We recommend using assays with extended incubation phases to monitor current HIV vaccine efficacy trials.

Epitopes corresponding to the envelope genetic subtype are present on the surface of free virions of HIV-1 group M primary isolates and can be detected in neutralization assays with extended incubation phases.

The hypothesis is that there are neutralizing epitopes on the surface of free virions of human immunodeficiency virus type 1 (HIV-1) that correspond to the genetic subtype of the envelope glycoprotein. Assays with extended incubation and reduced absorption phases are required to demonstrate neutralization with antibodies to these epitopes. These assays quantify virus infectivity, rather than reductions in release of antigen into culture supernatants. Neutralizing antibodies reduce virus infectivity by at least 80%, as scored by the presence/absence of antigen released after 14 days in culture of mitogen-transformed peripheral blood mononuclear cells (PBMCs). The epitopes are shared within different subtypes of group M, but not group O, isolates. Individual plasma, selected from three, independent panels of seropositive individuals, cross-neutralize within each subtype as well as the combinations of A with C, B with D or G, and C with CRF01_AE. Isolates within subtype B show the greatest variation in their resistance to neutralization, ranging from highly sensitive to highly resistant. No highly sensitive subtype D isolates were identified. Isolates from subtypes A, C, and CRF01_AE were all resistant. The strategic implication for vaccine design is that antibodies to a limited number of epitopes can neutralize more than 90% of the HIV-1 isolates that are circulating currently in the world. Also, since only antibodies that produce an all-or-nothing loss in virus infectivity can reasonably be expected to prevent the viremic phase after in vivo infection, assays with extended incubation, and culture phases should be used to monitor current efficacy trials.

Inter-subtype cross-neutralizing antibodies recognize epitopes on cell-associated HIV-1 virions.

HIV-1 infected individuals with cross-neutralizing antibodies against primary HIV-1 isolates belonging to Group M (envA-H) and O, are identified. To investigate the neutralization-kinetics of primary isolates with these antibodies, different neutralization assay conditions are compared. Each set is summarized as a/b/c where a is the time in hours for which antibody is incubated with virus, b is the time in hours allowed for virus to absorb to cells, c is the total culture period in days, from the cells' first exposure to virus, before antigen production (peripheral blood mononuclear cells) or number of fluorescent cells (GHOST) are measured. In HIV-infected individuals, neutralizing antibodies can be detected against a wide range of primary isolates (Group M; A-H and Group O) in PBMC-assays with short incubation phases (1/2/7 or 1/24/7). If cultures are extended (1/2/14 or 1/24/14), however, neutralization can be lost. In kinetic experiments, neutralization can even be seen without pre-incubation (a=0 hr). This study shows that neutralization of primary HIV isolates by cross-reactive antibodies can continue after the virus has bound to its target cell. This neutralization, however, is not an all or nothing loss in virus infectivity. Most often it leads only to a reduction in viral replication rates.