Reprogramming the antigen specificity of B cells using genome-editing technologies

Voss, James E.; González-Martín, Alicia; Andrabi, Raiees; Fuller, Roberta P.; Murrell, Ben; McCoy, Laura E; Porter, Katelyn; Huang, Deli; Li, Wenjuan; Sok, Devin; Le, Khoa; Briney, Bryan; Chateau, Morgan; Rogers, Geoffrey L.; Hangartner, Lars; Feeney, Ann J.; Nemazee, David; Cannon, Paula M.; Burton, Dennis R.

doi:10.7554/elife.42995

Cited by 75 publications

(73 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In contrast, B cell receptor reprogramming in primary cells using retroviruses has not been successful 39 . Moreover, although antibody heavy chains have been targeted into human B cells using CRISPR/Cas9 40 , little is known about how CRISPR/Cas9 genome targeting might be used to introduce complete antibody genes into mature B cells that retain the ability to participate in immune responses in vivo .…”

Section: Discussionmentioning

confidence: 99%

Antigen-specific humoral immune responses by CRISPR/Cas9-edited B cells

Hartweger

McGuire

Horning

et al. 2019

Preprint

View full text Add to dashboard Cite

24A small number of HIV-1 infected individuals develop broadly neutralizing-antibodies to 25 the virus (bNAbs). These antibodies are protective against infection in animal models. 26However, they only emerge 1 -3 years after infection, and show a number of highly 27 unusual features including exceedingly high levels of somatic mutations. It is therefore 28 not surprising that elicitation of protective immunity to HIV-1 has not yet been possible. 29Here we show that mature, primary mouse and human B cells can be edited in vitro 30 using CRISPR/Cas9 to express mature bNAbs from the endogenous Igh locus. 31Moreover, edited B cells retain the ability to participate in humoral immune responses. 32Immunization with cognate antigen in wild type mouse recipients of edited B cells elicits 33 bNAb titers that neutralize HIV-1 at levels associated with protection against infection. 34This approach enables humoral immune responses that may be difficult to elicit by 35 traditional immunization. 363 Main text 37Although a vaccine for HIV remains elusive, anti-HIV-1 bNAbs have been identified and 38 their protective activity has been demonstrated in animal models [1][2][3][4] . These antibodies 39 are effective in suppressing viremia in humans and large-scale clinical trials to test their 40 efficacy in prevention are currently under way 2, 5-12 . However, these antibodies typically 41 have one or more unusual characteristics including high levels of somatic hypermutation 42 (SHM), long or very short complementarity determining regions (CDRs) and self-43 reactivity that interfere with their elicitation by traditional immunization. 44 45 Consistent with their atypical structural features, antibodies that broadly neutralize HIV-46 1 have been elicited in camelids, cows and transgenic mice with unusual pre-existing 47 antibody repertoires [13][14][15][16][17][18] . However, even in transgenic mice that carry super-physiologic 48 frequencies of bNAb precursors, antibody maturation required multiple immunizations 49 with a number of different sequential immunogens. Moreover, bNAbs only developed for 50 one of the epitopes targeted 15, 17, 18 . Consequently, elicitation of bNAbs in primates or 51 humans remains a significant challenge. 52 53To bypass this issue, we developed a method to reprogram mature B cells to express 54 an anti-HIV-1 bNAb. Adoptive transfer of the engineered B cells and immunization with 55 a single cognate antigen led to germinal center formation and antibody production at 56 levels consistent with protection. 57 58 4 Results 59 Expressing antibodies in primary mature, murine B cells. 60To edit mature B cells efficiently, they need to be activated and cultured in vitro. To 61 determine whether such cells can participate in humoral immune responses in vivo we 62 used Igh a CD45.1 B cells carrying the B1-8 hi heavy chain that are specific for the hapten 63 4-hydroxy-3-nitro-phenylacetyl (NP) 19 . B1-8 hi B cells were activated in vitro with anti-64 RP105 antibody for 1 -2 days and subsequently transferred into c...

show abstract

Section: Discussionmentioning

confidence: 99%

Antigen-specific humoral immune responses by CRISPR/Cas9-edited B cells

Hartweger

McGuire

Horning

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…The gene sequences of HIV bnAbs and other desirable antibodies can however be engineered into the genomes of ex vivo activated primary B cells, such that they are expressed as functional B cell antigen receptors (BCRs) using endogenous heavy chain (HC) constant genes 3–5 . As such, engineered BCRs can undergo class switching for eventual secretion as protective antibodies from plasma cells.…”

Section: Figurementioning

confidence: 99%

“…Current engineering strategies can result in the expression of self-reactive BCRs due to pairing of engineered Ig chains with those endogenously produced by the targeted cell 3–5 . While tolerance mechanisms in the periphery of mice and humans generally ensure that autoreactive B cells are non-functional 22–25 , we sought to ensure that this would still be the case for cells activated ex vivo using the methods we required for efficient HDR based B cell genome editing.…”

Section: Figurementioning

confidence: 99%

“…However, their elicitation is made difficult by low frequencies of appropriate precursor B cell receptors and the complex maturation pathways required to generate bnAbs from these precursors 2 . Antibody genes can be engineered into B cells for expression as both a functional receptor on cell surfaces and as secreted antibody 3–5 . Here, we show that HIV bnAb-engineered primary mouse B cells can be adoptively transferred and vaccinated in immunocompetent wild-type animals resulting in the expansion of durable bnAb memory and long-lived plasma cells.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Vaccine Elicitation of HIV Broadly Neutralizing Antibodies from Engineered B cells

Huang

Tran

Olson

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

24HIV broadly neutralizing antibodies (bnAbs) can suppress viremia and protect against 25 chronically infected patients shows that they generally derive from B cell precursors with uncommon 42 antigen receptor features such as long third heavy chain complementarity determining regions 43 (CDRH3s) which then require extensive somatic hypermutation 8,9 . 44 45 The gene sequences of HIV bnAbs and other desirable antibodies can however be engineered into 46 the genomes of ex vivo activated primary B cells, such that they are expressed as functional B cell 47 antigen receptors (BCRs) using endogenous heavy chain (HC) constant genes 3-5 . As such, 48 engineered BCRs can undergo class switching for eventual secretion as protective antibodies from 49 plasma cells. B cells engineered in this way have been shown to confer protective levels of pathogen-50 specific antibody in vivo for several weeks following adoptive transfer into immunocompromised 51 hosts 5 , or for several days following transfer into immunocompetent hosts 4,5 . 52 53Long-term expression of HIV bnAbs generated from engineered B cells, which could be boosted 54 through vaccination and which could mature in affinity against relevant viral sequences in 55 immunocompetent hosts, has potential as an attractive functional HIV cure strategy, given that 56 bnAbs administered passively in the context of infection have been shown to suppress viremia, kill 57 infected cells, and enhance host immunity 1,10,11 . Wild-type (WT) black 6 (C57BL/6J) mice represent 58 a useful model in which such an engineered B cell vaccine could be developed because of 59 similarities between mouse and human humoral immune systems, and because the mouse antibody 60 repertoire is also strongly genetically restricted in its ability to elicit HIV bnAbs [12][13][14] . In activated 61 primary B cells, we used CRISPR-Cas9 to insert the VRC01 HIV bnAb 15,16 light chain (LC) and HC 62 variable region (VDJ) into the mouse HC locus at J4 using a homology directed repair (HDR) genome 63 editing strategy. The inserted VRC01 gene is expressed under the control of a HC V-gene promoter 64 as a single mRNA, which is post-transcriptionally spliced to endogenous HC constant genes. A P2A 65 self-cleaving peptide sequence downstream of the mouse kappa (k) constant gene separates the 66 VRC01 light and heavy chains, allowing them to pair and form a functional cell surface expressed 67 BCR (H-targeting) (Fig. 1a). We chose to use the VRC01 genes because this prototype CD4 binding-68 site bnAb, which blocks entry of the virus into target CD4 + T cells, has been extensively tested in the 69 clinic for its ability to suppress viremia in patients and prevent infection after administration as a 70 recombinant monoclonal antibody 17-19 (clinicaltrials.gov NCT02568215, NCT02716675). 71 72A second strategy was also employed, in which the VRC01 heavy and kappa variable genes were 73 targeted separately to their endogenous loci for expression from V-gene promoter-controlled 74 transcripts spliced to cell-native ...

show abstract

“…Each developing B cell undergoes recombination of V, D, and J segments over more than a megabase of DNA within the heavy chain locus, and this results in variable regions that are essentially unique to each cell 16 . This sequence variability makes directly targeting antibody coding regions challenging, and replacement of the entire heavy chain locus has been inefficient to date 17 . To bypass this complexity, we developed a single cut approach where the full light chain linked to the heavy chain VDJ was inserted into an intronic region of the heavy chain locus.…”

mentioning

confidence: 99%

B cells engineered to express pathogen-specific antibodies using CRISPR/Cas9 protect against infection

Moffett

Fitzpatrick

et al. 2019

Preprint

View full text Add to dashboard Cite

Effective vaccines inducing lifelong protection against many important infections such as respiratory syncytial virus (RSV), human immunodeficiency virus (HIV), influenza and Epstein-Barr virus (EBV) are not yet available despite decades of research. As an alternative to a protective vaccine we developed a genetic engineering strategy in which CRISPR/Cas9 was utilized to replace endogenously-encoded antibodies with antibodies protective against RSV, HIV, influenza or EBV in primary human or murine B cells. The engineered antibodies were expressed in up to 59% of primary B cells under the control of endogenous regulatory elements, which maintained normal antibody expression and secretion.Importantly, a single transfer of murine B cells engineered to express an antibody protective against RSV resulted in potent and durable protection against RSV infection in immunocompromised hosts.This approach offers the opportunity to achieve sterilizing immunity against pathogens for which traditional vaccination has failed to induce or maintain protective antibody responses.

show abstract

Reprogramming the antigen specificity of B cells using genome-editing technologies

Cited by 75 publications

References 48 publications

Antigen-specific humoral immune responses by CRISPR/Cas9-edited B cells

Antigen-specific humoral immune responses by CRISPR/Cas9-edited B cells

Vaccine Elicitation of HIV Broadly Neutralizing Antibodies from Engineered B cells

B cells engineered to express pathogen-specific antibodies using CRISPR/Cas9 protect against infection

Contact Info

Product

Resources

About