Functional Improbable Antibody Mutations Critical for HIV Broadly Neutralizing Antibody Development

SUMMARYHIV-1 broadly neutralizing antibodies (bnAbs) require high levels of activation-induced cytidine deaminase (AID) catalyzed somatic mutations for optimal neutralization potency. Probable mutations occur at sites of frequent AID activity, while improbable mutations occur where AID activity is infrequent. One bottleneck for induction of bnAbs is the evolution of viral envelopes (Envs) that can select bnAb B cell receptors (BCR) with improbable mutations. Here we define the probability of bnAb mutations and demonstrate the functional significance of key improbable mutations in three bnAb B cell lineages. We show that bnAbs are enriched for improbable mutations, implying their elicitation will be critical for successful vaccine induction of potent bnAb B cell lineages. We outline a mutation-guided vaccine strategy for identification of Envs that can select B cells with BCRs with key improbable mutations required for bnAb development. Our analysis suggests that through generations of viral escape, Env trimers evolved to hide in low probability regions of antibody sequence space.

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Characterization of neutralizing antibodies from a SARS-CoV-2 infected individual

10.1101/2020.05.12.091298 ◽

2020 ◽

Cited By ~ 21

Author(s):

Emilie Seydoux ◽

Leah J. Homad ◽

Anna J. MacCamy ◽

K. Rachael Parks ◽

Nicholas K. Hurlburt ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Neutralizing Antibodies ◽

Therapeutic Potential ◽

Infected Individual ◽

Neutralizing Antibody ◽

Viral Envelope ◽

List Type ◽

Cell Lineages

ABSTRACTB cells specific for the SARS-CoV-2 S envelope glycoprotein spike were isolated from a COVID-19-infected subject using a stabilized spike-derived ectodomain (S2P) twenty-one days post-infection. Forty-four S2P-specific monoclonal antibodies were generated, three of which bound to the receptor binding domain (RBD). The antibodies were minimally mutated from germline and were derived from different B cell lineages. Only two antibodies displayed neutralizing activity against SARS-CoV-2 pseudo-virus. The most potent antibody bound the RBD in a manner that prevented binding to the ACE2 receptor, while the other bound outside the RBD. Our study indicates that the majority of antibodies against the viral envelope spike that were generated during the first weeks of COVID-19 infection are non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt the SARS-CoV-2 spike-ACE2 interaction can potently neutralize the virus without undergoing extensive maturation. Such antibodies have potential preventive/therapeutic potential and can serve as templates for vaccine-design.IN BRIEFSARS-CoV-2 infection leads to expansion of diverse B cells clones against the viral spike glycoprotein (S). The antibodies bind S with high affinity despite being minimally mutated. Thus, the development of neutralizing antibody responses by vaccination will require the activation of certain naïve B cells without requiring extensive somatic mutation.HighlightsAnalysis of early B cell response to SARS-CoV-2 spike proteinMost antibodies target non-neutralizing epitopesPotent neutralizing mAb blocks the interaction of the S protein with ACE2Neutralizing antibodies are minimally mutated

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In vitro affinity maturation of broader and more-potent variants of the HIV-1–neutralizing antibody CAP256-VRC26.25

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2106203118 ◽

2021 ◽

Vol 118 (29) ◽

pp. e2106203118

Author(s):

Yiming Yin ◽

Brian D. Quinlan ◽

Tianling Ou ◽

Yan Guo ◽

Wenhui He ◽

...

Keyword(s):

B Cell ◽

Posttranslational Modifications ◽

Neutralizing Antibodies ◽

Cytidine Deaminase ◽

Neutralizing Antibody ◽

Affinity Maturation ◽

Homology Directed Repair ◽

Activation Induced Cytidine Deaminase ◽

Hiv 1

Three variable 2 (V2) loops of HIV-1 envelope glycoprotein (Env) trimer converge at the Env apex to form the epitope of an important classes of HIV-1 broadly neutralizing antibodies (bNAbs). These V2-glycan/apex antibodies are exceptionally potent but less broad (∼60 to 75%) than many other bNAbs. Their CDRH3 regions are typically long, acidic, and tyrosine sulfated. Tyrosine sulfation complicates efforts to improve these antibodies through techniques such as phage or yeast display. To improve the breadth of CAP256-VRC26.25 (VRC26.25), a very potent apex antibody, we adapted and extended a B cell display approach. Specifically, we used CRISPR/Cas12a to introduce VRC26.25 heavy- and light-chain genes into their respective loci in a B cell line, ensuring that each cell expresses a single VRC26.25 variant. We then diversified these loci through activation-induced cytidine deaminase–mediated hypermutation and homology-directed repair using randomized CDRH3 sequences as templates. Iterative sorting with soluble Env trimers and further randomization selected VRC26.25 variants with successively improving affinities. Three mutations in the CDRH3 region largely accounted for this improved affinity, and VRC26.25 modified with these mutations exhibited greater breadth and potency than the original antibody. Our data describe a broader and more-potent form of VRC26.25 as well as an approach useful for improving the breadth and potency of antibodies with functionally important posttranslational modifications.

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Early Pro-Inflammatory Signal and T-Cell Activation Associate With Vaccine-Induced Anti-Vaccinia Protective Neutralizing Antibodies

Frontiers in Immunology ◽

10.3389/fimmu.2021.737487 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jue Hou ◽

Shuhui Wang ◽

Dan Li ◽

Lindsay N. Carpp ◽

Tong Zhang ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

Neutralizing Antibodies ◽

Cell Activation ◽

Molecular Mechanisms ◽

Neutralizing Antibody ◽

Activator Protein 1 ◽

Successful Vaccine ◽

Hiv 1 ◽

Core Genes

Both vaccine “take” and neutralizing antibody (nAb) titer are historical correlates for vaccine-induced protection from smallpox. We analyzed a subset of samples from a phase 2a trial of three DNA/HIV-1 primes and a recombinant Tiantan vaccinia virus-vectored (rTV)/HIV-1 booster and found that a proportion of participants showed no anti-vaccinia nAb response to the rTV/HIV-1 booster, despite successful vaccine “take.” Using a rich transcriptomic and vaccinia-specific immunological dataset with fine kinetic sampling, we investigated the molecular mechanisms underlying nAb response. Blood transcription module analysis revealed the downregulation of the activator protein 1 (AP-1) pathway in responders, but not in non-responders, and the upregulation of T-cell activation in responders. Furthermore, transcriptional factor network reconstruction revealed the upregulation of AP-1 core genes at hour 4 and day 1 post-rTV/HIV-1 vaccination, followed by a downregulation from day 3 until day 28 in responders. In contrast, AP-1 core and pro-inflammatory genes were upregulated on day 7 in non-responders. We speculate that persistent pro-inflammatory signaling early post-rTV/HIV-1 vaccination inhibits the nAb response.

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Functional development of a V3/glycan-specific broadly neutralizing antibody isolated from a case of HIV superinfection

eLife ◽

10.7554/elife.68110 ◽

2021 ◽

Vol 10 ◽

Author(s):

Mackenzie M Shipley ◽

Vidya Mangala Prasad ◽

Laura E Doepker ◽

Adam S Dingens ◽

Duncan K Ralph ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Viral Escape ◽

Single Infection ◽

Structural Studies ◽

Broadly Neutralizing Antibodies ◽

Hiv Vaccines ◽

Functional Development ◽

Broadly Neutralizing Antibody

Stimulating broadly neutralizing antibodies (bnAbs) directly from germline remains a barrier for HIV vaccines. HIV superinfection elicits bnAbs more frequently than single infection, providing clues of how to elicit such responses. We used longitudinal antibody sequencing and structural studies to characterize bnAb development from a superinfection case. BnAb QA013.2 bound initial and superinfecting viral Env, despite its probable naïve progenitor only recognizing the superinfecting strain, suggesting both viruses influenced this lineage. A 4.15 Å cryo-EM structure of QA013.2 bound to native-like trimer showed recognition of V3 signatures (N301/N332 and GDIR). QA013.2 relies less on CDRH3 and more on framework and CDRH1 for affinity and breadth compared to other V3/glycan-specific bnAbs. Antigenic profiling revealed that viral escape was achieved by changes in the structurally-defined epitope and by mutations in V1. These results highlight shared and novel properties of QA013.2 relative to other V3/glycan-specific bnAbs in the setting of sequential, diverse antigens.

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Reprogramming the antigen specificity of B cells using genomeediting technologies

10.1101/455402 ◽

2018 ◽

Cited By ~ 1

Author(s):

James E. Voss ◽

Alicia Gonzalez-Martin ◽

Raiees Andrabi ◽

Roberta P. Fuller ◽

Ben Murrell ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cytidine Deaminase ◽

Variable Region ◽

Neutralizing Antibody ◽

Surface Expression ◽

Affinity Maturation ◽

Cell Surface Expression ◽

Human Antibody ◽

Antigen Specificity

We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody, PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved anti-HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses.

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High-affinity, neutralizing antibodies to SARS-CoV-2 can be made in the absence of T follicular helper cells

10.1101/2021.06.10.447982 ◽

2021 ◽

Author(s):

Jennifer S. Chen ◽

Ryan D. Chow ◽

Eric Song ◽

Tianyang Mao ◽

Benjamin Israelow ◽

...

Keyword(s):

B Cell ◽

Antibody Production ◽

Neutralizing Antibodies ◽

Influenza A ◽

Neutralizing Antibody ◽

New Paradigm ◽

High Affinity ◽

Tfh Cells ◽

Follicular Helper ◽

Canonical Pathways

T follicular helper (Tfh) cells are the conventional drivers of protective, germinal center (GC)-based antiviral antibody responses. However, loss of Tfh cells and GCs has been observed in patients with severe COVID-19. As T cell-B cell interactions and immunoglobulin class switching still occur in these patients, non-canonical pathways of antibody production may be operative during SARS-CoV-2 infection. We found that both Tfh-dependent and -independent antibodies were induced against SARS-CoV-2 as well as influenza A virus. Tfh-independent responses were mediated by a population we call lymph node (LN)-Th1 cells, which remain in the LN and interact with B cells outside of GCs to promote high-affinity but broad-spectrum antibodies. Strikingly, antibodies generated in the presence and absence of Tfh cells displayed similar neutralization potency against homologous SARS-CoV-2 as well as the B.1.351 variant of concern. These data support a new paradigm for the induction of B cell responses during viral infection that enables effective, neutralizing antibody production to complement traditional GCs and even compensate for GCs damaged by viral inflammation.

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Rapid selection of HIV envelopes that bind to neutralizing antibody B cell lineage members with functional improbable mutations

10.1101/2021.01.04.425252 ◽

2021 ◽

Author(s):

Olivia Swanson ◽

Brianna Rhodes ◽

Avivah Wang ◽

Shi-Mao Xia ◽

Robert Parks ◽

...

Keyword(s):

B Cell ◽

Neutralizing Antibodies ◽

Cell Lineage ◽

Neutralizing Antibody ◽

Developmental Pathways ◽

Broadly Neutralizing Antibodies ◽

Minimal Subset ◽

Functional Features ◽

Selection Of

SummaryElicitation of broadly neutralizing antibodies (bnAbs) by an HIV vaccine will involve priming the immune system to activate antibody precursors, followed by boosting immunizations to select for antibodies with functional features required for neutralization breadth. The higher the number of mutations necessary for function, the more convoluted are the antibody developmental pathways. HIV bnAbs acquire a large number of somatic mutations, but not all mutations are functionally important. Here we identified a minimal subset of mutations sufficient for the function of the V3-glycan bnAb DH270.6. Using antibody library screening, candidate envelope immunogens that interacted with DH270.6-like antibodies containing this set of key mutations were identified and selected in vitro. Our results demonstrate that less complex B cell evolutionary pathways than those naturally observed exist for the induction of HIV bnAbs by vaccination, and establish rational approaches to identify boosting sequential envelope candidate immunogens.

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Expansion of SARS-CoV-2-specific Antibody-secreting Cells and Generation of Neutralizing Antibodies in Hospitalized COVID-19 Patients

10.1101/2020.05.28.118729 ◽

2020 ◽

Cited By ~ 1

Author(s):

Renata Varnaitė ◽

Marina García ◽

Hedvig Glans ◽

Kimia T. Maleki ◽

John Tyler Sandberg ◽

...

Keyword(s):

B Cell ◽

Specific Antibody ◽

Neutralizing Antibodies ◽

Cell Activation ◽

Neutralizing Antibody ◽

Antibody Responses ◽

B Cell Activation ◽

Immunological Parameters ◽

Antibody Secreting Cell ◽

Antibody Levels

AbstractCoronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 and has since become a global pandemic. Pathogen-specific antibodies are typically a major predictor of protective immunity, yet B cell and antibody responses during COVID-19 are not fully understood. Here, we analyzed antibody-secreting cell (ASC) and antibody responses in twenty hospitalized COVID-19 patients. The patients exhibited typical symptoms of COVID-19, and presented with reduced lymphocyte numbers and increased T cell and B cell activation. Importantly, we detected an expansion of SARS-CoV-2 nucleocapsid protein-specific ASCs in all twenty COVID-19 patients using a multicolor FluoroSpot assay. Out of the 20 patients, 16 had developed SARS-CoV-2-neutralizing antibodies by the time of inclusion in the study. SARS-CoV-2-specific IgA, IgG and IgM antibody levels positively correlated with SARS-CoV-2-neutralizing antibody titers, suggesting that SARS-CoV-2-specific antibody levels may reflect the titers of neutralizing antibodies in COVID-19 patients during the acute phase of infection. Lastly, we showed that interleukin 6 (IL-6) and C-reactive protein (CRP) concentrations were higher in serum of patients who were hospitalized for longer, supporting the recent observations that IL-6 and CRP could be used to predict COVID-19 severity. Altogether, this study constitutes a detailed description of clinical and immunological parameters in twenty COVID-19 patients, with a focus on B cell and antibody responses, and provides tools to study immune responses to SARS-CoV-2 infection and vaccination.

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ADCC-mediating non-neutralizing antibodies can exert immune pressure in early HIV-1 infection

PLoS Pathogens ◽

10.1371/journal.ppat.1010046 ◽

2021 ◽

Vol 17 (11) ◽

pp. e1010046

Author(s):

Dieter Mielke ◽

Gama Bandawe ◽

Jie Zheng ◽

Jennifer Jones ◽

Melissa-Rose Abrahams ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Immune Escape ◽

Viral Evolution ◽

Neutralizing Antibody ◽

Protective Role ◽

Viral Escape ◽

Immune Pressure ◽

Control Virus ◽

Kinetics Of ◽

Hiv 1

Despite antibody-dependent cellular cytotoxicity (ADCC) responses being implicated in protection from HIV-1 infection, there is limited evidence that they control virus replication. The high mutability of HIV-1 enables the virus to rapidly adapt, and thus evidence of viral escape is a very sensitive approach to demonstrate the importance of this response. To enable us to deconvolute ADCC escape from neutralizing antibody (nAb) escape, we identified individuals soon after infection with detectable ADCC responses, but no nAb responses. We evaluated the kinetics of ADCC and nAb responses, and viral escape, in five recently HIV-1-infected individuals. In one individual we detected viruses that escaped from ADCC responses but were sensitive to nAbs. In the remaining four participants, we did not find evidence of viral evolution exclusively associated with ADCC-mediating non-neutralizing Abs (nnAbs). However, in all individuals escape from nAbs was rapid, occurred at very low titers, and in three of five cases we found evidence of viral escape before detectable nAb responses. These data show that ADCC-mediating nnAbs can drive immune escape in early infection, but that nAbs were far more effective. This suggests that if ADCC responses have a protective role, their impact is limited after systemic virus dissemination.

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