Immunization against HIV-1 with Adoptive Cell Transfer of Engineered B cells: A Review
Abstract
After 30 years into the race of making an HIV-1 vaccine, genome engineering technology such as CRISPR has enabled targeted therapeutic and adoptive cell transfer of engineered B cells. The engineering of B cell receptors (BCR) to express the broadly neutralizing antibodies (bNAbs) against HIV-1 can elicit a potent and durable humoral immunity against the viral infection which has been hard to create with traditional vaccination strategies. This article reviews literature in the past 3 years and outlines the fundamental strategies and techniques used in these studies. Before adoptive cell transfer of engineered B cells can become a commercialized prophylactic or therapeutic strategy against HIV-1, more explorations need to be done in primate and animal models, the longevity of B cells needs to be studied and the number of dosages and B cells required must be determined with more studies. With more exploration and technological advancements, adoptive cell transfer of engineered B cells as a prophylactic or therapeutic strategy can become cheaper and accessible to the general public.
Disclaimer: this paper has not been peer reviewed yet.
The global HIV epidemic
According to estimations by the World Health Organization, approximately 36.7 million people in the world were living with HIV by the end of 2015. It’s estimated that 0.8% of adults aged 15–49 are infected with HIV. Although the scale of the epidemic varies globally, 70% of people living with HIV live in Sub-Saharan Africa; this is the region with the most infections globally. Although the number of infections in the United States was reduced by 19% between 2005 and 2014, 40 000 new infections occurred in 2015 and the total number of people living with HIV in the United States is at 1.2 million. Antiretroviral therapy has reduced transmission of the virus and is the current preventative solution. Out of all the people in the world living with HIV, 46% have access to antiretroviral therapy. By the time this article was written (2021), there are still no commercial vaccines for the prevention of HIV infections [1]
HIV-1 Structure
HIV-1, the more common HIV virus, accounts for around 95% of all infections worldwide [3]. The strains of HIV-1 are organized into four types: group M, group N, group O, and group P. group M is mostly responsible for the global HIV epidemic [4]. The HIV-1 is a lentivirus with a genome that encodes for 16 proteins. The core of HIV-1 which contains the viral genomic RNA and the replication enzymes RT and integrase (IN), is enveloped by a cone-shapes shell that is composed by the viral capsid (CA) protein. CA protein can assemble into rings of five or six protomers. Thus, the cone is predominantly formed of hexamers. The virus can subvert the human innate and adaptive immune system. The process of replication starts when the virus attaches itself to cell surface receptors and ends when the emerging particles mature into virions. HIV-1 usually infects CD4+ T cells and macrophage cells. A host cell is infected when the viral DNA goes through reverse transcription and integration into the host cell chromosomes [5].
Preventative vaccine challenges
A preventative vaccine against HIV-1 does not exist because first there are many strains that belong to different subtypes of HIV. These strains can differ in their envelop by up to 35% [7] Current HIV-1 candidate vaccines have a restricted neutralizing reach. They can’t neutralize all the HIV-1 viruses [8]. Thus, a vaccine must be created with an immunogen that can initiate immune response to the major strains of HIV. A possible solution would be the induction of broadly neutralizing antibodies (bNAbs). Anti-HIV-1 bNAbs can neutralize the major strains that are common in human transmission. [7]. More detail is given on bNAbs in the subsection “Adoptive cell transfer of engineered B cells as an alternative to vaccines”.
CRISPR/Cas9 System
The cost-efficient and rapid genome engineering technology CRISPR/Cas9 easily allows the modification of the genome of mammalian cells. The CRISPR/Cas9, originally the “immune system” of prokaryotes against phages and other invaders, can be used to knockout and add genes into the genome of humans and other mammals with a Cas endonuclease, a gRNA and a template RNA depending on the purpose of the modification. To this date, protection against infection by HIV-1 has been given to hematopoietic stem/progenitor cells (HSPCs) [9] and CD4+ T cells through the deletion of CCR5 with the CRISPR/Cas9 system [10]. CAR T cell therapy uses an HR template of a chimeric antigen receptor (CAR) to a T cell receptor (TCR) which programs the T cell to kill cancer cells with the selected antigen [11].
B Cell Receptors (BCRs)
The BCR is composed of a heavy chain and a light chain. There are two BCR isotypes in a mature B cell: IgM and IgD. Both BCR isotypes are composed of membrane immunoglobin (mIg). There is a structure of four immunoglobin domains in IgM and a structure of five in IgD in the heavy chain. B cells are activated when an antigen binds with the BCR [12]. When an antigen is recognized by the BCR, T cells help with the functional differentiation of naïve B cells into plasma cells, germinal center (GC) B cells and memory B cells [13].
Adoptive cell transfer of engineered B cells as an alternative to vaccines
Only a small number of individuals infected with HIV-1 develop bNAbs 1–3 years after infection and they show highly unusual features and high levels of somatic mutations [14]. When studied, the BCR of those chronically infected patients who expressed HIV bnAbs was characterized by a long third heavy chain complementarity determining regions (CDRH3s), and for broad neutralization, they require extensive somatic hypermutation [15, 16]. Furthermore, the natural elicitation of HIV bNAbs is difficult because there is a low frequency of individuals with the appropriate BCR and the maturation pathway that enables the generation of bNAbs from the BCRs [17]. Therefore, elicitation of HIV-1 immunity has not yet been possible [14].
Since 2017, support for efforts of an HIV vaccine that elicits bnAbs to provide sterilizing immunity against a challenging virus has been provided by experiments in macaque and humanized mouse models [18, 15]. A barrier in the effort of creating such an HIV-1 vaccine is the genetic limitation of the human repertoire of BCR from which antibodies arise. As a solution, antibody genes can be edited into B cells to be expressed as a BCR on the cell surface or as a secreted antibody. Desirable antibodies such as mature HIV bnAbs can be engineered into the genome of ex vivo activated primary B cells which can then be added to the human repertoire of BC, overcoming the problem of a genetic limitation. These engineered BCRs use endogenous HC constant genes [19, 20]. This allows the engineered BCRs to undergo class switching for eventual secretion as protective antibodies from plasma cells. B cells engineered this way have shown to create protective levels of pathogen-specific antibodies in vivo for several weeks following adoptive cell transfer into the immunocompetent mice. It was several days for immunocompetent wild type (WT) mice [20, 14]. Vaccine elicitation of HIV bNAbs from engineered B cells in immunocompetent hosts could also contribute to a cure for HIV because it has been demonstrated that bNAbs administered passively can suppress viremia, kill infected cells, and enhance host immunity [21, 22].
What makes B cells a good medium for eliciting protection against viral infections is because B cells are “living and evolving drugs”, as stated by Nahmad et al., 2020. This study showed that upon immunization of mice, adoptively transferred B cells engineered to express anti-HIV bNAbs home to germinal centers (GC) are expressed instead of the endogenous antibody and that they can differentiate into memory and plasma cells while undergoing class switch recombination (CSR). GCs and CSR rates are accumulated when immunized with a high affinity antigen. When the immunization was boosted, the rate of edited B cells in GCs and antibody secretion increased, indicating memory retention. The sequencing of the antibody in engineered B cells in the spleen show pattern of clonal selection. Consequently, immunization against viral infections with B cells is a potential strategy that can be employed for protection against HIV-1 [23].
Chimeric B Cell Receptors (CBCR)
A chimeric B cell receptor (CBCR) is a synthetic BCR that is genetically engineered to express a specific antigen-binding site. Normally, in the case of an endogenous BCR, the antigens that bind to the surface of B cells depend on the variable region of the endogenous heavy and light chain. However, in CBCRs, the genetic modification of the variable region allows specified antigen binding, which means that B cells can be genetically engineered to elicit neutralization against specific viruses.
A study by Moffett et al., 2019 edited primary human B cells with CRISPR/Cas9 to express antibodies targeting respiratory syncytial virus (RSV), HIV, influenza virus, and Epstein-Barr virus (EBV). This study was not focused on HIV. The exploration was done in the presence of endogenous regulatory elements which maintained normal antibody expression and secretion, and thus the expression of the edited antibody was efficient. Protection against RSV infection was then tested in RAG1-deficient mice by a single transfer of edited B cells expressing an antibody against RSV. Potent and durable protection was demonstrated. Hence, this study sets the background for further explorations that are more focused on HIV [20].
Another study that identified general strategies for B cell editing to express a desired antibody is one by Greiner et al., 2019. This study demonstrated the expression of a desired monoclonal antibody in primary human B cells through the creation of CBCRs with CRISPR [24]. A monoclonal antibody (adalimumab) that neutralizes TNF-α was delivered at the CXCR4 locus and integrated into the genome of the B cell through CRISPR-mediated homology directed repair (HDR). Consequently, the heavy and light chain of the monoclonal antibody was expressed on the surface of the B cell from endogenous promoters. The HDR in the genome of the B cell was achieved by the activation of the B cell pre- and post-transfection. The genome editing was confirmed with tracking of indels by decomposition (TIDE) analysis of Sanger sequencing data and by CRISPR genome analyzer (CRISPR-GA) analysis of MiSeq data. Yet the MiSeq data showed that the efficiency of editing was low, and the B cells were less viable after activation and electroporation. Further validation and quantification of nanobody/antibody expression by ELISA with the soluble target antigen were indicated by the team. Furthermore, the study identified solutions to some of the challenges that first need to be overcome in order to create a clinical adoptive cell transfer therapy. For example, the hurdle of B cell differentiation into plasma cells can be overcome by exposure to particular cytokines [25] or via transfection with appropriate transcription factors [26]. Therefore, CBCRs that express a specific antigen-binding site can be achieved with HDR yet the editing efficiency of CRISPR, B cell viability, and B cell differentiation are still challenges that must be addressed before a clinical-stage therapy.
Another study in the same year by Pesch et al., 2019 outlined a methodology that can potentially overcome some of the challenges imposed by the Greiner et al., 2019 study. This methodology describes how molecular design can be optimized and how CBCRs can be more efficiently integrated into the genome of Murine B cells. The final outcome of the study was the integration of several cassettes into the genome of primary murine B cells and immortalized hybridoma B cells through CRISPR-Cas9 induced HDR. To analyze the murine B cell’s surface expression of HEL-binding and Strep-tag II detection, flow cytometry was used, which demonstrated the surface expression and antigen recognition of the CBCR. It was observed that to increase the surface expression of the CBCR, pre-activation of the B cells and the inclusion of Strep-tag II and CD79β Signaling Domain was beneficial. These B cells equipped with CD79β signalling domain are envisioned to proliferate into memory and plasma cells upon exposure to the antigen, creating long-term humoral immunity in the host. Additionally, although HDR limitations were observed in murine B cells, the same cannot be seen in human B cells, thus, it is suggested that there are differences in DNA repair mechanism in human B cells in comparison with murine B cells, rendering human B cells a more viable medium for adoptive immune cell therapy. Consequently, to improve the integration of CBCRs into the genome of Murine B cells, pre-activation of the B cells and the inclusion of Strep-tag II and CD79β Signaling Domain would be a proposed solution [27]. Nevertheless, this study did not specify how the challenges of B cell differentiation can be addressed.
HIV Broadly Neutralizing Antibodies
Since endogenous BCR genes can be genetically engineered to express CBCRs on the surface of B cells, the question remains if CBCRs that express HIV bnAbs can be engineered. A handful of such studies have been done in the past 5 years. These studies further develop engineering, transfection and differentiation methods for the engineering of HIV bnAb-expressing B cells.
In 2019, Voss et al., demonstrated the genome engineering of mature human B cells to express the HIV bnAb, PG9 through VDJ replacement by HDR. The immunoglobulin (Ig) gene of the 3 donor B cells was modified by HDR to replace the heavy-chain variable region of B cell with an HIV bnAb. Previous studies have shown that antibodies contain most of the code for protective paratopes in their heavy chain [28, 29, 30, 31]. The engineered heavy chain is then paired with an endogenous light chain to form a functioning CBCR [32]. They chose to continue with the development of their engineering strategies using the PG9 heavy chain paratope in Ramos B cells because the PG9 heavy chains pair well with diverse LCs (including the functional Ramos light chain), even though there is a loss of neutralizing breadth with some light chains. gRNAs with the highest efficiencies were found using the pCAG-eGxxFP recombination assay in 293 cells. To respond to the challenge of genomic rearrangement during the genesis of B cells, they introduced the double stranded DNA breaks after the most 5’ V-gene promoter (V7–81) and after the most distant J gene (J6) because HRs 5’ and 3’ to these sites are the same in every B cell. In human primary polyclonal B cells that have undergone a diversity of VDJ recombination, they used the V3–74 promoter; this resulted in higher engineering efficiency. Through coverage depth analysis, they found that many double strand breaks were repaired with NHEJ. To reduce or eliminate NHEJ in future experiments, the authors suggest creating a donor DNA format adapted to skew repair towards HDR. The class switching of the immunoglobin from the endogenous to the PG9 gene was done through activation-induced cytidine deaminase (AID). This method was employed on cells that had undergone VDJ recombination with any combination of variety (V), diversity (D) and joining (J) genes. AID was specifically advantageous because it maintained the affinity maturation of the B cells during the adaptive immune response. This is because AID retains the genetic flexibility of the cell. The 3 donors had at least 10 folds more cells with the PG9 CBCR. Sequenced mRNA showed that the PG9 heavy chain resulted in multiple isotypes after culture with CD40 ligand and IL-4. Finally, the engineered B cells could bind to the PG9 epitope [19]. Therefore Voss et al., 2019 propose a comprehensive editing strategy that outlines a cell culturing method, a DNA cutting strategy that does not interfere with genomic recombination and a method that maintains class switching of the immunoglobin. Although the editing strategy is comprehensive, this study only edited the heavy chain.
Although Voss et al., 2019 targeted the antibody heavy chains of human B cells using CRISPR/Cas9, much is still not known about introducing complete antibody genes into mature B cells with CRISPR/Cas9 that will be able to fully participate in immune responses in vivo. In response, Hartweger et al., 2019 developed a method to edit the endogenous Igh locus of mature, primary mouse and human B cells were using CRISPR in vitro to express mature bNAbs. The method involves short-term culture in vitro, silencing of endogenous Ig genes, and insertion of a bi-cistronic cDNA into the Igh locus. Off-target double stranded breaks and integrations were avoided by limiting culture time which prevented the growth of these cells and by using nonviral gene editing with ssDNA templates to limit random integrations. The allelic exclusion was maintained by ablating the igkc gene in order to prevent the expression of multiple antibodies and additional chimeras that can impede the efficiency of humoral immunity and by eliciting unwanted autoimmunity. Furthermore, not using a promoter increased surface BCR expression and improved safety. Still, expression of the edited BCR for different antibodies varied, similar to antibody transgene in mice, however, the expression levels were enough to induce antibody production from antigen exposure in vivo. The authors have noted that their neutralization measurements may be an underestimate because bNAbs produced an IgM or isotypes other than IgG were excluded. Finally, they found that the edited B cells could still participate in humoral immune responses because when the wild-type mice who had received the edited B cells were exposed to the cognate antigen, the bNAb titers were elicited and they neutralized the HIV-1 at the level of protection against the infection [14]. This study achieved a humoral immune response that Is difficult to obtain with traditional immunization.
Similarly, a study by Huang et al., 2020 attempted the adoptive cell transfer and vaccination of HIV bNAb-engineered primary mouse B cells into immunocompetent mice. They showed the creation of durable bnAb memory and plasma cells that lived for a long time. More specifically, this study showed that HIV bNAb-engineered primary B cells can be expanded and affinity-matured in vivo through vaccination which results in durable bNAb memory and long-lived plasma cells in wild-type mice. To measure neutralization, Elisa and virus neutralization assays were used to measure total antigen-specific and engineered antibody responses in the serum. Mice that received mock engineered or H + κ targeted cells elicited the lowest total antigen-specific and VRC01-competitive antibody response. Whereas 10-fold higher total antigen-specific titers were seen in mice that received H-targeted cells after the first boost which was accompanied by significantly higher levels of VRC01-competitive antibody in the serum. As well, memory and plasma cell expansion were monitored. Only mice that received H-targeted cells showed high frequencies of VRC01-engineered GC B cells. splenic and bone marrow plasma cells and MCs two weeks after the first boost. Thus, immunized VRC01-B cells matured into GC-dependent memory and plasma cells. Finally, it was seen that somatic hypermutation after immunization allowed the edited B cells to adapt to an evolving pathogen — which is the case with HIV-1 [17]. Being one of the most recent works in the field of B cell editing to express HIV bNAbs, this study by Huang et al., 2020 is an accumulation of previous explorations and a good baseline to look at for future scientific and technological advancements.
Discussion
The success of editing at the BCR loci and expression of the defined monoclonal antibody suggests a therapeutic approach through reprogramming of the B cell’s antigen specificity such as cellular humoral vaccines or adoptive cell transfer for evolved pathogens [24, 19]. An adoptive cell therapy using B cells would be favoured for their longevity and protein secretory capacity. The humoral immune response produced consists of durable and potent production of neutralizing antibodies, thus overcoming the challenges of current vaccines [27]. This protective and self-tolerant antibody response can safely and efficiently happen in primary B cells by autologous re-engraftment through vaccination [19]. The creation of durable bNAb memory and long-lived plasma cells and hypermutation that allows adaptation to evolving pathogens are all markers that make engineered B cells a potential solution towards protection against viral infections and contribute towards making an HIV cure [17].
To test the feasibility of edited B cells for prophylactic or therapeutic use in humans or even as a tool to enhance infectious disease experimental model systems, more exploration needs to be done in animal models first [19]. Some other further explorations to be done should be experimentation in primate models to determine if millions of edited B cells are required to achieve a therapeutic effect in the patient, experimentation to understand the longevity of the antibody response produced by edited B cells and if optimization by boosting or other methods is required [14].
Given the current scientific advancement, presently, adoptive cell therapies are expensive, but they will become cheaper with future scientific developments. Edited B cells serve as an especially good HIV prophylactic strategy because most protective vaccine responses such as neutralizing antibody responses depend on humoral immunity, which is already elicited for most human pathogens, but it has not been possible to do so for HIV-1. On a greater spectrum, using adoptive cell transfer to elicit humoral immunity can be applied to any disease requiring a specific antibody response [14].
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