B cell targeting by molecular adjuvants for enhanced immunogenicity

ABSTRACT

Introduction

Adjuvants are critical components of vaccines to improve the quality and durability of immune responses. Molecular adjuvants are a specific subclass of adjuvants where ligands of known immune-modulatory receptors are directly fused to an antigen. Co-stimulation of the B cell receptor (BCR) and immune-modulatory receptors through this strategy can augment downstream signaling to improve antibody titers and/or potency, and survival in challenge models.

Areas covered

C3d has been the most extensively studied molecular adjuvant and shown to improve immune responses to a number of antigens. Similarly, tumor necrosis superfamily ligands, such as BAFF and APRIL, as well as CD40, CD180, and immune complex ligands can also improve humoral immunity as molecular adjuvants.

Expert opinion

However, no single strategy has emerged that improves immune outcomes in all contexts. Thus, systematic exploration of molecular adjuvants that target B cell receptors will be required to realize their full potential as next-generation vaccine technologies.

KEYWORDS:

• Immune modulation
• adjuvant
• c3d
• tumor necrosis family receptors
• baff
• april
• cd40
• cd180
• immune complexes
• b cell

1. Introduction

Vaccines are invaluable tools to prevent infection and halt the spread of diseases [1]. The availability and fast production of effective and safe vaccines to emerging pathogens have been highlighted in recent months due to the ongoing COVID-19 pandemic. Unfortunately, effective vaccines against many prominent pathogens remain elusive despite researchers’ best efforts. While live attenuated vaccines remain one of the most effective forms of vaccination, concerns of potential virulence in susceptible individuals and reversal of attenuation often preclude their development. As a result, recombinant subunit vaccines have emerged as an alternative due to their greater safety and tolerability, since these rely solely on isolated components of pathogens and are incapable of causing infection in the host [2]. A key advance in the development of subunit vaccines has been the identification of antigens from the sequencing of pathogens, made possible by vast improvements in sequencing technologies. Leveraging this technology, recombinant proteins mimicking antigens of interest in their native form can be produced, aiming to induce long-lived immunological memory primarily through the elicitation of antibodies toward the protective epitopes of the antigen [3,4], as was demonstrated for the recently licensed meningococcus B vaccine [5]. Thus, a new generation of subunit vaccines takes advantage of the identification of key antigenic epitopes from a pathogen.

Importantly, almost all current vaccines rely on the presence of antibodies circulating in serum or on mucosa that block infection or bacteremia/viremia, and these often represent the best correlate of vaccine-induced protection [1,6,7]. Correspondingly, advances in sequencing technologies have also enabled the characterization of antibody responses to many antigens, parsing out the characteristics of desirable immune responses that should be preferentially elicited by immunization. As such, eliciting the highest titer of protective antibodies that focus the response on the most protective epitopes of antigens often constitutes a primary goal of vaccination [8,9]. For instance, one area of high interest is the mapping of potent epitopes of the Plasmodium falciparum circumsporozoite protein (CSP) for the design of next-generation vaccines against malaria. Recent reports have identified the central ‘NANP’ repeat region of CSP and sequence motifs upstream of this repeat as recognized by potent inhibitory antibodies [10–13], and suggested its flanking C- and N-terminal domains may not contain as potent neutralizing epitopes [14,15]. Indeed, understanding non-neutralizing antibody responses can also provide important insights into antigen design: by avoiding such epitopes, the elicitation of antibodies that have poor protective function can be minimized to favor protective antibody responses.

Beyond antigen sequence, the structural requirements underlying antigen recognition by potently protective antibodies has been extensively studied. For instance, the surface-expressed trimeric fusion machinery of multiple viruses have been shown to require stabilization in a pre-fusion state to elicit antibodies that provide potent protection (e.g. Respiratory syncytial virus (RSV), human immunodeficiency virus (HIV), and severe acute respiratory coronavirus 2 (SARS-CoV-2) [15–25]). A third consideration for antigen design against pathogens with vast genetic diversity, such as HIV and influenza, is the elicitation of broadly neutralizing antibodies (bnAbs), which are able to neutralize virus despite sequence heterogeneity between viral strains [26]. This can often be achieved by focusing the response on epitopes conserved across strains and minimizing antibodies against variable regions within the antigen [27–30]. One approach to elicit bnAbs is ‘germline-targeting,’ in which epitopes included in the immunogen are selected to engage specific germline precursor B cells, thus leveraging the naturally occurring process of affinity maturation to shepherd the immune system toward the generation of bnAbs. This strategy is being employed by the HIV vaccine candidate eOD-GT8, where the outer domain of gp120 was engineered to engage the inferred germline precursor of the VRC01 antibody [31]. Here, the modified gp120 led to improvements in the breadth and affinity of the VRC01-like antibody response when used as a prime, and boosted with more native trimeric spikes [31–33]. Thus, as discussed above, the purposeful selection of antigenic epitopes, as well as their mode of presentation, are considerations of high importance to promote protective antibody responses and minimize off-target, non-neutralizing antibodies against challenging pathogens.

Despite improvements in antigen design, recombinant subunit vaccines are notorious for their poor immunogenicity. Strategies to promote and support immune responses elicited by a subunit vaccine are thus a critical consideration in vaccine design. Traditional adjuvants have proven to be excellent tools to enhance the immunogenicity of vaccine candidates by reducing the number of doses required to achieve protection [34], prolonging memory elicited by the vaccine [35,36], and expanding the breadth and potency of elicited antibodies [37]. Currently, several adjuvants are approved for use in humans. These adjuvants vary in composition (e.g. aluminum salts, TLR agonists, emulsions, etc.), and most of them have been shown to engage the innate immune system to ultimately enhance the magnitude of adaptive responses [38–40] through a variety of mechanisms [37,39,41–43]. The choice of adjuvant has been shown to significantly impact immune outcomes resulting from vaccination, which can range from complete protection to poor immunogenicity and no protection for the same antigen [44]. This is exemplified by the RTS,S malaria vaccine, which is based on Plasmodium falciparum CSP: initial studies of this vaccine adjuvanted with an aluminum salt (alum) was successful in eliciting anti-CSP antibodies in the sera, but conferred no protection [45]. When RTS,S was administered together with an oil-in-water emulsion adjuvant (AS02A) or liposomal formulation (AS01B), 30–50% protection was observed [46]. Thus, the choice of a suitable adjuvant for a given vaccine can be pivotal, and has been an area of intense research (reviewed in [47–49]).

Beyond the clear benefits provided by antigen optimization and adjuvants targeting innate immunity to augment protective responses, could the specific targeting of receptors on lymphocytes provide additional adjuvant effects? In this review, we describe recent efforts in the design of molecular adjuvants particularly targeting B cell receptors for the enhancement of immunogenicity in vaccination.

1.1. B Cell Signaling Upon Antigen Encounter

Antibody-mediated immunity orchestrated by B cells plays a vital role in the protection against pathogens. The recognition of antigen by a highly specific B cell receptor (BCR) displayed on the surface of a B cell initiates signaling processes and is termed ‘Signal 1’ (depicted in Figure 1A). The interaction between antigen and BCR induces receptor micro-clustering on the cell surface, triggering signaling cascades via the phosphorylation of key intermediary kinases (such as PKC, Akt, and MAPK) and influx of calcium, resulting in the transcriptional activation of NFAT and NFκB [50–52]. These signaling processes leading to downstream B cell activation are tightly regulated by antigen specificity and affinity [53,54], as well as by co-receptors clustering with the BCR; these can act in concert with the BCR to either stimulate (e.g. CD19, CD21) or inhibit (e.g. CD22, CD45, FcγRIIb) the activation signal [55–59]. Following Signal 1, B cells process the antigen and present antigenic fragments on Major Histocompatibility Complex (MHC) class II molecules to cognate T cells primed by the same antigen earlier in the response, enabling them to receive additional signals via costimulatory molecules upregulated on both cell types after priming (‘Signal 2’) (e.g. CD80/86, CD40, ICOS-L) (Figure 1B) (reviewed in [60]). These signals induce B cells to rapidly proliferate and form structures called germinal centers (GCs) within secondary lymphoid organs (SLOs), where somatic hypermutation (SHM) and class-switch recombination (CSR) take place to yield a higher-affinity, multifunctional antibody response [61]. A third set of signals (‘Signal 3’) provides essential survival and developmental cues to B cells; these are usually maintained by soluble mediators within the cell’s microenvironment, such as BAFF, APRIL and interleukin-4 (IL-4) (Figure 1C) [62–65]. Following activation, B cells undergo many cell fate decisions. They can differentiate into plasma cells (PCs) specialized in the production and secretion of antibodies targeting the antigen [66]; most of these are short-lived within the SLO and rapidly undergo apoptosis, but a subset of PCs migrate to the bone marrow, where their longevity enables them to contribute over 80% of the antibody present in serum [67–69]. Alternatively, B cells can differentiate into memory cells, poised to quickly react upon re-exposure to the antigen. In this case, memory B cells can reenter the GC reaction to undergo further SHM, or differentiate into PCs at this later time point [70]. B cell fate is regulated via receptors present on its surface at multiple checkpoints (signals 1, 2 and 3). Thus, targeting B cell co-receptors at each of these signals could be a strategic avenue to modulate immune responses to vaccination. Several receptors on the surface of B cells have been shown to be capable of altering immune responses following antigen interaction with the BCR. Of these, the modulation of B cell signaling via CD21, a co-receptor known to lower the threshold for B cell activation and enhance BCR signaling, has been most extensively studied. The inhibitory co-receptor FcγRIIb has also been studied, conversely known to dampen BCR signaling and cellular activation. Other receptors with more general roles in overall B cell survival and differentiation (e.g. signals 2 and 3), such as the tumor necrosis factor (TNF) superfamily members have also been actively researched. This review explores these receptors for their potential to be targeted by molecular adjuvants with the goal to modulate immune responses and alter cellular outcomes that improve recombinant vaccines.

Figure 1. Overview of B cell signaling. A) Signal 1 – antigen interacts with the B cell receptor (BCR) on the surface of B cells to initiate a signaling cascade leading to the downstream activation of transcription factors NFAT, NFκB and AP-1 for the activation of B cells. The signal initiated by the antigen-BCR interaction is strictly regulated by stimulatory (i.e. CD21, CD19) and inhibitory (i.e. CD22, FcγRIIb) receptors micro-clustering with the BCR upon antigen recognition. B) Signal 2 – antigen is internalized, processed, and presented on the B cell on MHC class II molecules to cognate T cells primed earlier in the response, enabling additional signals from T cells to promote B cell proliferation, CSR and SHM. C) Signal 3 – interactions of TNFSF receptors with soluble mediators in the microenvironment (BAFF, APRIL, IL-4) promote B cell survival and suppress apoptosis. Created with Biorender.com

2. C3d

The complement system is essential for immune homeostasis and plays a critical role to bridge the innate and adaptive arms of the immune system. One critically important member of the complement system is C3d, which is derived from complement component 3 (C3) and results in C3d-tagged micro-organisms or antigens (reviewed in [71,72]) with the capacity to bind to complement receptor 2 (CR2 or CD21) on B cells and follicular dendritic cells for opsonization, as well as to assist in antigen presentation and the maintenance of immunological memory [73]. CD21 is typically found on the surface of B cells in association with CD19 and CD81 [74,75] and plays an integral role in immune regulation (reviewed in [76,77]). While CD21 itself does not have a signaling motif, binding of CD21 to C3d results in downstream signaling through the immunoreceptor tyrosine-based activation motifs (ITAMs) on CD19, resulting in improved B cell survival and proliferation [78–80]. When C3d-tagged pathogens simultaneously interact with CD21 and the BCR, pathway crosstalk decreases the signal threshold of antigen required to trigger B cell activation [81]. This pathway crosstalk is also capable of reducing inhibitory signals and preventing apoptosis, which are important factors for the induction of immunological memory.

There is a long history of using C3d as a molecular adjuvant to boost B cell signaling in response to various antigens in a vaccination setting. First demonstrated by Dempsey et al. in 1996, a 10,000-fold increase in secondary immune responses was obtained for 2–3x murine C3d (mC3d) tagged hen egg-white lysozyme (HEL) in comparison to HEL alone [81]. Since this initial finding, similar results have been achieved for viral, bacterial, parasitic and self-antigens, as summarized in Table 1 (and reviewed in [72,82]). Fusion of tandem repeats of C3d (three is often sufficient) to antigens and administration through various routes results in improved antibody titers, often also improving neutralization and survival in challenge experiments in mice. Interestingly, C3d fusion seems most frequently to polarize the immune system toward a Th2 response in these vaccinated mice, characterized by IgG1 dominance and the secretion of high levels of IL-4 and IL-5 [83–86], although Th1-dominated responses have also been observed [87].

Table 1. Summary of immunization experiments containing C3d

Pathogen	Antigen	C3d derivative	Vaccine type	Animal model	Administration route	Conclusion	Reference	Year
– –	Hen egg lysozyme (HEL)	1–3x murine C3d (mC3d)	Protein	Mice	Subcutaneous (S.C.) prime, intraperitoneal (I.P.) boost	Over 10,000-fold enhancement of antibody titers for 2x and 3x mC3d fusions.	[81]	1996
Influenza	sHA	3x mC3d	DNA	BALB/c mice	Gene gun	Fusion of secreted hemagglutinin (sHA) to 3x C3d accelerated affinity maturation of resulting antibodies and appearance of hemagglutinin-inhibition activity. C3d fusion resulted in complete protection from virus challenge at a 10-fold lower dose in comparison to HA alone.	[144]	2000
Influenza	sHA from two strains (A/Puerto Rico/8/34 (H1N1) and A/Aichi/2/68-x31 (H3N2)	3x mC3d	DNA	BALB/c mice	Gene gun	Mice immunized with 3x C3d-sHA showed enhanced protection against heterologous viruses but lacked neutralization capability and strong IL-4 cytokine release.	[88]	2003
Bovine Rotavirus, Bovine Herpes virus	BRV VP7 BHV-1 gD	1–2x mC3d	DNA	C57BL mice	Gene gun or I.P.	No enhancement of antibody responses; resulted in significantly lower number of IFN-γ secreting cells for both antigens.	[91]	2001
Measles	Hemagglutinin	3x mC3d	DNA	BALB/c mice	Gene gun	Hemagglutinin (H)-3C3d fusion induced increased titers of anti-H antibodies with higher neutralization capable of reducing measles plaque formation.	[89]	2001
HIV-1	gp120	3x mC3d	DNA	BALB/c mice	Gene gun	gp120-mC3d immunized mice showed higher antibody titers to gp120 and faster onset of affinity maturation in comparison to gp120 alone.	[145]	2001
HIV-1	gp120	1–3x mC3d, then 3x hC3d	DNA prime, protein boost	BALB/c mice, New Zealand white rabbits	Mice – Gene gun prime, intramuscular (I.M.) boost Rabbits – I.M. prime and boost	2x and 3x mC3d fused gp120 boosted antibody titers and affinity maturation in comparison to gp120 and gp120-C3d but was unable to rescue low levels of neutralizing antibody responses.	[90]	2003
HIV-1	gp120	3x mC3d	DNA	C57BL/6, BALB/c, C3H/He, CD-1 Swiss mice	Gene gun	Responses in inbred mice were primarily IgG1, responses in outbred strain was a mix of IgG1 and IgG2a. Cytokine responses were IL-4 and IFN-γ dominant suggesting mixed Th response, affinity maturation was enhanced in outbred mice.	[87]	2004
HIV-1	gp120	4x P28 or 3x mC3d	DNA or Protein	BALB/c mice	DNA – Gene gun, I.M. or intranasal (I.N.) Protein – I.M. or I.N.	Both gp120-P28 and gp120-mC3d induced higher titers and better antibody avidity in comparison to gp120 controls in both DNA and protein immunization experiments regardless of immunization route.	[94]	2006
Malaria	PbCSP (-A)	3x mC3d	DNA	BALB/c mice	Gene gun	Reduced protection and suppression of IgG1 responses due to 3x mC3d fusion. Furthermore, induction of antigen-specific, IL-4 producing splenocytes was also supressed due to 3x mC3d fusion.	[92]	2005
Malaria	PbCSP (-A)	3x P28	DNA	BALB/c mice	Gene gun	Enhanced immunogenicity and protection in comparison to PbCSP(-A) and PbCSP(-A)-P28; predominant Th2 response. Enhanced clinical protection at 6-week in C3d-immunized groups suggests enhanced memory responses in these mice in comparison to controls.	[83]	2007
Malaria	PbMSP142 and PfMSP142	N-terminal or C-terminal 3x mC3d	DNA	BALB/c mice	Gene gun	mC3d-PfMSP142 and PfMSP142-C3d showed earlier onset of higher titers of MSP142 specific IgG1 antibodies but were unable to prevent parasite growth in vitro and did not enhance the production of IL-4 or IFN-γ producing T cells In a P. berghei challenge model, mC3d-PbMSP142 and PbMSP142-mC3d did not enhance antibody titers at the experiment endpoint. Challenge with P. berghei showed no significant delay in parasitemia.	[98]	2010
Diphtheria	Diphtheria toxin fragment B	3x mC3d	Protein	CBA/J, C3H/HeJ mice	I.P.	Reduced antibody responses in comparison to DT-B alone.	[93]	2006
– –	hCGβ	3x mC3d	DNA	BALB/c mice	I.M.	C3d-fused hCGβ groups had more rapid onset of anti-hCG titers with higher overall titers at the end of the immunization regimen in comparison to hCGβ alone groups. hCGβ-C3d groups had higher levels of IL-4 and IL-10 but decreased IFN-γ and IL-2, suggesting a C3d-mediated bias toward Th-2 responses.	[86]	2006
– –	Amyloid β1-11	3x C3d + PADRE	DNA	C57BL/6 mice	Gene gun	C3d fusion to Aβ1-11-PADRE constructs enhanced antibody titers in comparison to Aβ1-11 alone. Humoral immune responses were biased toward a Th2-type with the IgG1/IgG2a^b ratio significantly higher in the C3d group. Furthermore, Aβ1-11-C3d elicited significantly greater antigen-specific CD4 + T cell proliferation and IL-4/IL-5 cytokine production in comparison to Aβ1-11 alone, further confirming a Th-2 bias.	[85]	2008
West Nile Virus	Glycoprotein E, Ecto E, prM/E	2x P28	DNA	C57BL/6 mice	I.M. or gene gun	Conflicting results depending on the immunogen and route of delivery, i.e. gene gun results in best titers while prM/E is consistently the most protective immunogen despite lack of P28 fusion. However, P28-mediated enhancement of antibody titers is observed for DIII with I.M. administration and at lower doses via gene gun administration.	[95]	2010
Porcine reproductive and respiratory syndrome virus	GP5	P28 (2x, 4x, 6x)	DNA	BALB/c mice	I.M.	P28 fused GP5 increased antibody titers regardless of number of P28 repeats (2x, 4x, 6x) in comparison to GP5 alone, but titers were lower for all samples in comparison to inactivated vaccine controls. P28 fused constructs showed increased levels of IL-4 and IFN-γ, suggesting both a Th-1 and Th-2 type response. Immunization with GP5-P286x resulted in significantly higher levels of these cytokines in comparison to other P28 repeats and GP5 alone but the inactivated vaccine resulted in the highest levels of induced IL-4 and IFN-γ production.	[97]	2011
Salmonella enterica serovar Enteritidis	FimA	mC3d (1–3x)	Protein	BALB/c mice	S.C. prime, I.P. boost	Improved immune protection from Salmonella enterica serovar Enteritidis lesions with addition of sequential C3d (50% protection with no C3d, 100% protection with 3 copies).	[146]	2012
Trichinella spiralis	Attenuated Salmonella enterica expressing surface or secreted Ag30	3x P28	Attenuated Salmonella transformed with plasmid antigen	BALB/c mice	I.N.	Ag30-P28 group showed increasing IgA titers over timeand improved protection from T. spiralis challenge characterized by high amounts of IgG1 and IL-5 production, suggesting a Th2 response.	[84]	2014
Rabies	Glycoprotein G linear epitope G5	1x P28	DNA	BALB/c mice	I.M.	P28 fusion resulted in sustained neutralizing antibody response over 90 days (waned in G alone immunogens), correlating with improved protection and clinical scores.	[96]	2018

Table 2. Summary of immunization experiments containing TNFSF ligands. Ligands from rabbit sequences are denoted with an ‘r’ and murine ligands are denoted with an ‘m.’

Pathogen	Antigen	Ligand	Vaccine type	Animal model	Administration route	Conclusion	Reference	Year
HIV	gp120 and Env (SOSIP.R6-IZ-D7324)	rAPRIL, rBAFF, rCD40L	DNA prime, protein boost	New Zealand White Rabbits	Gene gun S.C. prime; intradermal (I.D.), I.M. and S.C. boost n	All three fusions increased AID expression and secretion of IgM, IgG and IgA in vitro. Env-APRIL induced higher anti-Env antibody titers and heterologous tier 1 neutralization.	[125]	2012
HIV	Gag	mBAFF mGITRL mCD27L m4-1BBL mIL-12p70	Adenoviral vector	BALB/c mice	I.M., I.P., I.V.	Mice immunized with Ad5 gag vectors co-administered with soluble SP-D scaffolded BAFF, GIRTL, CD27L and 4-IBBL and IL-12p70 constructs as adjuvants were challenged with vaccinia-Gag. BAFF and 4–1BBL containing immunizations resulted in significantly reduced replication in comparison to controls, where IL-12, CD27L, and D-GITRL were not protective.	[141]	2014
HIV	Gag, gp140	mAPRIL mBAFF	DNA	BALB/c mice	I.M.	Co-immunization with SP-D scaffolded multi-trimers of BAFF and APRIL induced neutralizing antibodies against tier 1 and 2 viruses, as well as increased germinal center activity, antibody secreting cells and anti-gp120 functional avidity.	[126]	2015
Rabies	rRABV	mAPRIL	Live-attenuated RABV	C57BL/J6 mice	I.M.	No effect in anti-RABV titers and did not improve the generation of long-lived plasma cells against RABV.	[123]	2017
Rabies	rRABV	mBAFF	Inactivated recombinant RABV	C57BL/J6 mice	I.M.	Induced faster and higher titers of neutralizing antibodies without affecting the longevity of the antibody response.	[122]	2019
Influenza	H5N1 HA VLP	mAPRIL mBAFF	VLPs	BALB/c mice	I.M.	Both BAFF-VLP and APRIL-VLP showed significantly higher antibody titers in comparison to VLP alone. BAFF-VLP induced production of more potent/broad neutralizing antibodies.	[124]	2019

It is well established that multiple copies of C3d fused to the antigen of interest is required for optimal adjuvant effect. However, in some cases, C3d fusion has been shown to reduce the efficacy of the immunogen [91–93]. One possible explanation for this effect is that C3d fusion to the antigen may block important epitopes on the antigen, making them inaccessible for the development of antibody responses. Indeed, one such example was suggested when the malaria immunogen Plasmodium berghei CSP (PbCSP) was fused to 3x tandem repeats of mC3d at its C-terminus [92]. DNA immunizations in BALB/c mice resulted in a reduction of anti-CSP antibody titers and loss of protective efficacy in a challenge model of infection in comparison to PbCSP alone [92]. This issue was resolved in a follow-up study through the fusion of 3x tandem repeats of the minimal CD21 binding domain of mC3d, termed P28, in place of full-length mC3d. This smaller fragment of mC3d rescued mC3d-mediated enhancement and resulted in improved antibody titers, enhanced immunogenicity, and improved protection in comparison to both CSP alone and CSP-mC3d fusions [83].

This is one example of many where using P28 as a molecular adjuvant has been shown to be similarly robust or even superior to mC3d for CD21-mediated enhancement of immune responses to a given antigen. Recent studies have employed P28 in DNA vaccines targeted toward HIV [94], West Nile [95] and Rabies [96] viruses, as well as in an attenuated Salmonella vaccine candidate that expresses or secretes the Trichinella spiralis antigen Ag30 [84], amongst others [85,97]. These studies report a P28-mediated enhancement of antibody titers and Th2 bias, but was often dependent on the antigen and immunization route employed. For example, in the case of West Nile virus immunogens, high antibody titers were observed upon gene gun administration for all tested antigens (prM/E, EctoE, DIII) regardless of the presence of P28, whereas upon intramuscular (I.M.) administration with the same antigens, higher antibody titers were observed when EctoE or DIII was fused to P28. Interestingly, EctoE-P28 administered via gene gun was the only antigen in this study (other than prM/E – the positive control) that was able to provide complete protection against West Nile challenge [95]. These results further highlight the importance of both immunogen design and the exploration of different administration routes for optimal vaccine efficacy. In a different study, the use of attenuated Salmonella expressing T. spiralis antigen Ag30 was explored either alone or fused to P28 via a C-terminal linker before being attached to the surface of Salmonella (either by a cleavable or non-cleavable linker). The highest antibody titers and neutralization potency were obtained when Ag30-P28 was attached to attenuated Salmonella via a cleavable linker. In fact, the same antigen fused to P28 through a non-cleavable linker behaved poorly in immunizations in comparison to cleavable Ag30-P28, as well as Ag30 alone [84]. This study also suggested that the position of the P28 fusion is critical; an optimal adjuvant effect was only obtained when P28 was located at the C-terminus of the antigen. This observation is consistent with other studies that reported optimal enhancements where C3d or P28 were fused to antigens at the C-terminus [94,97,98].

The wealth of data available for C3d/P28-mediated enhancement of humoral immune responses supports CD21 targeting as an avenue for the improvement of vaccine candidates, although the magnitude of the adjuvant effect appears to depend strongly on the antigen, administration route, animal model, and vaccine type. In addition, our incomplete understanding of the mechanism of action of C3d-linked antigens was highlighted following the results of Haas et al. in 2004, which showed C3d-mediated enhancement of immune responses in CD21^-/-/CD35^-/- mice [99]. This data raises important questions about C3d specificity and the mechanisms of action of immune modulation via C3d. As a result, alternative mechanisms have been proposed for the observed immune enhancement, including :1) improved antigen half-life through C3d fusion, 2) additional availability of T cell epitopes that may be present on C3d, and 3) alternate C3d receptors that may facilitate immune modulation other than CD21 and CD35 [99]. Further studies looking into the biological impact of C3d fusion at the cellular level and in immune signaling are required to fully understand the effect of C3d fusion in these systems and represent an important avenue to assess whether molecular adjuvants can have an impact on other aspects of a successful vaccine candidate, such as improved memory and long-lived plasma cell (LLPC) populations.

3. Tumor necrosis family (TNF) receptors

The tumor necrosis factor (TNF) superfamily (TNFSF) of ligands and their receptors play critical roles for cellular survival, proliferation, and affinity maturation. The TNFSF is composed of 19 ligands and 29 receptors, which have been reviewed extensively for their roles in cell death, survival, proliferation, and differentiation [100,101]. Of the TNF ligands, B cell Activating Factor (BAFF, also known as BLyS) and A Proliferation Inducing Ligand (APRIL) represent attractive targets for their ability to modulate immune outcomes through B cell targeting, although additional TNFSF members have been explored for their co-stimulatory effects on dendritic cells and T cells as well (reviewed in [102]). BAFF is a critical B cell survival ligand, without which normal B cell maturation cannot occur [103]. Conversely, while APRIL is known to play a key role in the survival of LLPCs in the bone marrow, APRIL-deficient mice do not show any developmental or immune cell defects, suggesting that APRIL is not essential for normal immune development and function [104]. BAFF and APRIL are both type II membrane proteins expressed primarily by T cells, macrophages, dendritic cells, and monocytes. Like many other ligands in the TNFSF, proteolytic cleavage of BAFF and APRIL at their conserved furin cleavage site results in the release of active, soluble ligands. BAFF can be found in its membrane active form, but cleavage to form soluble, extracellular BAFF is essential for B cell homeostasis. Indeed, mutation of the BAFF furin cleavage site results in immune dysfunction and impairment of antibody responses [105]. APRIL, however, is cleaved intracellularly in the Golgi prior to release and is typically only found in its soluble form extracellularly [106]. BAFF and APRIL are most commonly found in circulation as homotrimers [107], however, unlike other TNFSF members, BAFF has also been found as a 60-mer in physiological conditions [108]. Interestingly, APRIL, but not BAFF, is capable of binding heparin sulfate proteoglycans (HSPGs) [109], a function which may assist in the clustering and concentration of APRIL on a local cell surface, similar to the avidity effect that could be obtained by 60-mer BAFF binding to multiple receptors on a single cell [110].

BAFF and APRIL are both capable of interacting specifically and with high affinity with either B-Cell Maturation Antigen (BCMA), or Transmembrane Activator and Cyclophilin ligand Interactor (TACI). BAFF also has additional specificity to BAFF receptor (BAFF-R) and the recently described Nogo-66 receptor (NgR) [111]. BCMA, TACI, and BAFF-R are type I transmembrane proteins that are expressed primarily by B cell lineages at varying levels depending on the stage of B-cell development (summarized in [110]). BAFF-R is expressed on immature B cells following the expression of a functional BCR. The interaction between BAFF and BAFF-R is critical for the development and homeostasis of mature B cells and acts as a primary driving force in B cell survival and maturation. Similar to the phenotypes observed in BAFF-deficient mice, BAFF-R-deficient or -mutated mice show a block in B cell maturation ultimately resulting in autoimmunity and other immune defects [112,113]. Following the initial interactions of BAFF with BAFF-R in early B cell maturation, BAFF and APRIL play key roles in interacting with TACI and BCMA on mature B cell populations. TACI is highly expressed on marginal zone B cells and B1-B cells where it plays key roles in T cell-independent responses, inhibition of B cell expansion [114,115], IgG to IgA class switching, and the differentiation and survival of PCs [116,117]. BCMA expression is restricted to plasmablasts and PCs, and its role is primarily limited to the survival of LLPCs in the bone marrow. BCMA binds with high affinity to APRIL and lower affinity to BAFF, although this low affinity can be modulated through avidity effects, which have been observed via BCMA dimerization [118].

Another member of the TNFSF is CD40L, which is expressed primarily on CD4⁺ T cells [119]. CD40L, like BAFF and APRIL, can be proteolytically cleaved to create soluble forms of CD40L that retains full biological activity, though CD40L is more commonly considered a membrane-bound ligand [120]. CD40L interacts directly with CD40 on B cells and dendritic cells and plays critical roles in the provision of a T-cell help signal required in GC formation and for antibody isotype switching and affinity maturation [121]. Due to the roles of BAFF-R, TACI, BCMA, and CD40 in B cell differentiation and survival, they present themselves as targets for immune modulation in the context of vaccine design.

BAFF and APRIL homotrimers have both been utilized as molecular adjuvants for the improvement of immune responses toward challenging pathogens, as summarized in Table 2. In immunization studies for rabies virus, RABV viral vector immunogens expressing membrane-anchored BAFF have been shown to be capable of inducing faster and higher titers of neutralizing antibodies toward RABV antigens without adversely affecting the longevity of the immune response [122]. Conversely, in a separate study, anchoring of APRIL to RABV viral vectors had no effect on antibody responses, and APRIL co-engagement did not improve the generation of LLPCs [123]. These studies are some of the first to assess whether the addition of molecular adjuvants can affect the longevity of the induced memory response through the generation of LLPCs. This represents an avenue for further research to improve the resulting memory responses through this immunogen design platform and holds the potential to have significant impact for pathogens in which long-term memory remains a significant barrier in vaccine development, such as rabies and malaria. In influenza, WT, BAFF- and APRIL-containing H5N1 VLPs were utilized to immunize BALB/c mice in the presence of either alum or MPL adjuvants [124]. Both BAFF- and APRIL-VLPs showed significantly higher antibody titers in comparison to WT VLPs but only BAFF-VLPs were capable of improving the potency and breadth of the resulting neutralizing antibodies. In all cases, adjuvants were still required to achieve high titers and potency despite the presence of BAFF and APRIL as molecular adjuvants [124]. However, the longevity of the resulting memory responses remains to be assessed.

For HIV immunogen design, trimeric Env was C-terminally fused to either APRIL, BAFF, or CD40 in an attempt to target Env directly to B cells and improve Env-specific antibody responses [125]. For all three immunogens, there was an enhanced expression of Activation-induced cytidine-deaminase (AID) in comparison to controls in vitro upon incubation with antigen-specific B cells, suggesting the potential for an improved level of SHM through TNFSF co-engagement. Env-CD40L was the most effective in boosting AID expression in this system. In all three cases, the immunogens were capable of inducing IgM, IgG, and IgA secretion from human B cells in vitro. However, in an immunization experiment in rabbits, only Env-APRIL was capable of inducing high titers of Env-specific antibody responses with heterologous tier 1 neutralization in vivo [125]. In a more recent study, BALB/c mice were co-immunized with a mixture of DNA plasmids containing Env gp140 together with multi-trimers of BAFF or APRIL (TNFSF trimers multimerized on surfactant protein-D (SP-D) scaffolds) and IL-12, followed by a protein boost with Env. It is important to note that for this study, Env, BAFF/APRIL multi-trimers and IL-4 were all present on different plasmids and were not fused to each other. Despite not being directly fused, this immunization strategy was found to induce neutralizing antibodies against tier 1 and 2 viruses. The APRIL multi-trimer-containing vaccine candidate was particularly effective at generating tier 2 neutralization. Furthermore, both BAFF and APRIL candidates were capable of inducing enhanced GC reactivity, as well as increased anti-gp120 antibody-secreting cells and improved anti-gp120 avidity [126].

These results together show that the use of BAFF, APRIL, and CD40L as molecular adjuvants to specifically target BAFF-R, BCMA, TACI, and CD40 on B cells is a promising approach to improve vaccine outcomes against various pathogens. Interestingly, similar to the responses observed for C3d fusion, careful antigen design and systematic assessment of the different molecular adjuvants seems to be essential for enhancement of immune responses by these TNFSF ligands. For immunization schemes with both rabies and influenza antigens, BAFF-fused antigens showed the best overall immune responses, with increased titers and enhanced neutralization potency by resulting antibody responses, but were unable to improve the generation of effective immunological memory through increased LLPCs. Conversely, for HIV antigens, APRIL-fused Env resulted in the most favorable and reproducible antibody responses across two different studies, and direct fusion of APRIL to Env was not required for the adjuvant effect to be observed. However, the longevity of this response was not assessed. This data highlights the importance of a systematic evaluation of the use of TNF ligands as molecular adjuvants and stresses the value of testing multiple adjuvants in parallel, as a single strategy that works for all antigens hasn't yet been identified.

4. CD180

CD180 is a TLR receptor expressed both on B cells and DCs and plays a critical role in regulating the responses of these cell types to TLR ligands. On B cells, it has been shown that CD180 is important for B cell activation, and that directly targeting this receptor with an anti-CD180 antibody can trigger CD180-induced calcium flux, B cell activation, and proliferation [127,128]. Furthermore, internalization following ligation has been described for CD180, making it an attractive target for immunogen design, as CD180 internalization can assist with antigen processing by DCs and B cells to activate T cells in signal 2 [129]. CD180 is closely related to TLR4 and is an orphan member of the TLR family [130], which are known as effective targets for adjuvants [38,42]. Due to these factors, CD180 represents an intriguing target as a molecular adjuvant to improve immune responses.

In a recent study, authors used an anti-CD180 (αCD180) monoclonal antibody directly fused to the antigen hapten 4-hydroxy-3-nitro-phenacetyl (NP) to act as molecular adjuvant to boost immune responses [129]. In comparison to NP fused to an isotype control and αCD180 alone (without NP antigen), NP-αCD180 induced a 15-fold increase in polyclonal serum IgG titers and strong antigen-specific IgG and IgM immune response in WT, CD40 KO, and T cell-deficient mice. Furthermore, in comparison to NP in alum, NP-αCD180 induced a rapid anti-NP IgG response which peaked 11 days sooner than NP in alum in WT mice. NP-αCD180 was also shown to be effective in inducing GC formation, affinity maturation, and immunological memory. This platform was then expanded to assess the effect of CD180 targeting with the protein antigen ovalbumin (OVA). Strong Ag-specific IgG responses were observed in the OVA-αCD180 group in comparison to OVA in alum controls and OVA-isotype controls. These results were dependent on the direct fusion of αCD180 to the antigen of interest, as co-administration of unfused antigen with αCD180 did not result in a robust immune response [129].

In a follow-up study, the authors showed that CD180 targeting of OVA and HEL antigens induced rapid BCR internalization and that all B cell subsets were subsequently able to process and present the internalized antigens for CD4⁺ T cell help in WT mice [131]. These results were expanded to explore APC generation in BAFF-R^−/- mice, and the authors were able to demonstrate that αCD180, but not αCD40 targeting induced efficient antigen processing and presentation, even in the immature B cell subsets present within these mice. Within 48 h of immunization, αCD180 targeting induced the maturation of T follicular helper (T_fh) cells, suggesting that this targeting strategy is effective for the development of robust T-dependent humoral responses [131].

These studies are the first to specifically target CD180 as a molecular adjuvant and employ a novel technique of using an αCD180-specific monoclonal antibody – as opposed to the natural ligand of the receptor – for co-engagement. Follow-up studies that expand the breadth of antigens tested to include pathogens of high-priority will be required to fully understand the possible utility of CD180 as a molecular adjuvant in vaccination.

5. Immune complexes

There is over a century of history describing the use of immune complexes (ICs) in vaccination [132–134]. The earliest experiments in humans involved complexing live vaccines with anti-sera to improve both the safety and efficacy of the earliest vaccine candidates [132,135]. The generation of ICs involves the complexation of immunogens of interest with specific monoclonal or polyclonal antibodies. These antibodies are then capable of interacting with Fcγ receptors on immune cells (macrophages, monocytes, DCs, B cells, etc.) promoting efficient antigen uptake and processing, providing improved T cell help for the generation of immune memory and antibody responses [136–138].

One implication of IC vaccination is the specific interaction with Fcγ receptors on B cells that may modulate the outcome of B cell responses. A recent study showed that immune responses toward the seasonal trivalent inactivated influenza vaccine (TIV) could be modulated through the complexation of the TIV with purified polyclonal Abs from individuals that had a high sialic acid prevalence in their antibodies [139]. Immunization with these complexes (but not complexes made with asialylated IgGs) showed improved neutralization capacity, despite lower titers. The proposed model to explain these results was that ICs with sialylated IgG bound specifically to CD23 on B cells, resulting in the upregulation of the inhibitory cell-surface Fc receptor FcγRIIb. This upregulation of FcγRIIb would then increase the signal threshold required for B cell activation, thus limiting the cells that are activated to only those that possess high cognate BCR affinity and specificity to the antigen. In turn, this would improve the quality, but not necessarily the magnitude, of the B cell response [139].

This study suggests an intriguing mechanism for modulation of the quality of antibody responses at Signal 1 on B cells. Nonetheless, the specific interaction between sialylated IgGs and CD23 on B cells remains to be fully demonstrated biophysically. A recent study investigated this specificity but was unable to detect binding of IgG to CD23 or DC-SIGN through cellular surface plasmon resonance imaging (cSPRi) or by fluorescence activated cell sorting (FACS) regardless of the glycosylation state of the antibody [140]. These studies represent an interesting avenue of research that requires further investigation to determine the ability of an increased signal threshold on B cells to improve the quality of antibody responses. If successful, this approach could have considerable implications across fields where poor antibody quality and neutralization capacity continue to plague efforts toward an effective vaccine.

6. Expert opinion

Co-engagement of the BCR with immune-modulatory co-receptors is an approach with several successful examples of improving immunization outcomes, including improved antibody titers, potency, breadth and/or improved survival in challenge experiments [81,122,124,139,141]. The majority of these experiments have been performed in mouse models, with a select few in rabbits. Expanding animal models to include those closest to humans (i.e. non-human primates) would provide critical insight into the translatability of these molecular adjuvant approaches across model organisms, toward the goal of being able to use these as vaccination strategies in humans, which has not yet been attempted.

Through the direct fusion of antigens to natural ligands that target either stimulatory (CD21, BCMA, BAFF-R, CD180) or inhibitory (FcγRIIb and TACI) receptors on B cells, the outcome of the immunological responses of vaccination can be altered [84,86]. Unfortunately, there does not seem to be a single molecular adjuvant, vaccine type, or immunization route that is capable of consistently boosting immune responses across all antigens. However, this is not an unfamiliar problem in the vaccine field, as vaccine formulation, adjuvant composition and immunization route often require optimization for the best possible immune outcomes, even in classical vaccine designs [142]. This, however, suggests that despite the promise of molecular adjuvants to modulate immune responses, it is not a‘plug-and-play’ system that can be universally deployed. Thus, optimization is required to identify the most efficacious signaling pathway to target for an antigen of interest. Consequently, it is of critical importance that studies employing these molecular adjuvants continue to do so in a systematic way to properly assess the best strategy for immunogen design, as is common for chimeric immunogens [143]. Assessing best methods for conjugating molecular adjuvants to antigens should also be explored to ensure that the chimeric immunogens are designed in a way that optimizes both protective epitopes on the antigen and functional binding sites for the molecular adjuvant. Indeed, it has been shown in the context of C3d that the terminus to which the adjuvant is fused as well as its size can be critical for the quality of the resulting immune responses [83,84,92]. In addition, expanded studies to look at the different types of molecular adjuvants (i.e. C3d, TNFSF, CD180, and ICs) within the same study would be valuable for the assessment of the best molecular adjuvants for a given antigen.

Some datasets covered in this review contain conflicting results, which could in part be due to the promiscuous nature of ligand binding to B cell co-receptors. Indeed, it is well established that many of the natural ligands used in these studies have targets on B cells, as well as on other cell populations. As such, it becomes difficult to deconvolute with certainty which pathway is associated with the observed modulation of immunity. For C3d, antibody enhancement was evident even in CD21^-/- mice, suggesting additional mechanisms of action to the one initially envisioned [78]; immune complexes can bind Fcγ receptors on heterogenous populations of immune cell types [138]; and the family of TNF receptors and their ligands are notoriously promiscuous in their binding capabilities [100,101]. One approach to remove this complexity is to use an alternate, more specific molecule for targeting immune receptors of interest. Antibodies, or antigen-binding fragments (Fabs) of antibodies, serve as an excellent alternative to natural ligands and have been proven to be effective as a molecular adjuvant in CD180-targeting strategies [129,131]. Expanding this platform to include antibodies that are specific to receptors targeted by these molecular adjuvants (CD21, BCMA, BAFF-R, TACI, CD40) could potentially be key to understanding the mechanisms of immune modulation through B cell co-receptor targeting. Indeed, the improved specificity provided by antibodies would ensure that only the receptor of interest is being engaged by a chimeric immunogen.

Receptor clustering has been repeatedly shown to be critical for immunogens employing the use of molecular adjuvants. Targeting of CD21 through C3d requires at least 2–3 repeats of C3d or P28 for enhancement [81,83,94,97,144–146], whereas targeting through the TNFSF requires that ligands are presented in trimeric arrangement for receptor engagement [124–126] or even multimerization into multi-trimers using an SP-D scaffold [126,141]. The use of protein-based nanoparticles for adjuvant multimerization represents an attractive avenue for improved B cell modulation [147]. Naturally-occurring protein-based nanoparticles, such as ferritin [30,148], lumazine synthase [31,33], E2p [149] as well as non-natural two-component protein nanoparticles, such as I53-50 [150], could be utilized to co-display antigens of interest and molecular adjuvants. Using these nanoparticles for this function would ensure avidity effect for the antigen, thus improving BCR clustering, while also allowing for multiple copies of the molecular adjuvant to be presented simultaneously. Investigation of the symmetry features of these protein-based nanoparticles can also guide immunogen design to be able to include higher-order ligands, such a TNFSF homotrimers at three-fold axes within the nanoparticles [30,148–150].

Expanding these experiments will provide valuable insight into the best strategies for immune modulation via molecular adjuvants, and also help uncover previously unknown features of B cell biology. Indeed, these experiments offer the opportunity to closely examine the downstream signaling pathways associated with the targeting receptors (Figure 1) and expand our understanding of how their modulation impacts the expansion of the immune repertoire. Importantly, the design of next-generation vaccines against challenging pathogens will require novel strategies to induce long-lived protective antibodies and potentially the quick re-activation of the memory compartment to outpace pathogens. Whether this can be achieved by the molecular adjuvants presented in this review, among other strategies, remains to be determined. An improved understanding of basic B cell biology at the molecular level will provide promise for the emergence of hypotheses in immune modulation and translate into next-generation adjuvant technologies.

Article Highlights

• Molecular adjuvants can act by simultaneously targeting the B cell receptor and immune-modulatory receptors to augment downstream signaling cascades and improve immune responses in immunizations.

• Tandem repeats of C3d, the natural ligand of complement receptor 2 (CR2 or CD21) on B cells, and P28, a minimal binding domain of C3d, have been extensively studied as molecular adjuvants.

• Members of the tumor necrosis family of ligands (BAFF-R, TACI, BCMA) represent a key class of immune-modulatory receptors that have been targeted by molecular adjuvants through the fusion of their natural ligands with antigens.

• CD180 has recently emerged as a target of molecular adjuvants. Targeting CD180 with an αCD180 monoclonal antibody showed enhancements in antigen internalization and processing, as well as antibody titers for select model antigens.

• Although the enhancement of immune responses by immune complexes is well established, the exact mechanism of this approach for immune modulation of B cells remains to be fully elucidated.

• No single molecular adjuvant has been shown to be superior for all antigens, immunization routes and animal models. Thus, systematic studies continue to be required for each immunogen towards the goal of being able to use these types of molecular adjuvants in a clinical setting.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Author contributions

The text was written and edited by TS, AK and JPJ. The figure was designed and made by AK.

Acknowledgments

We would like to thank Bebhinn Treanor and Michael Ratcliffe for their input and discussions.

Additional information

Funding

T Sicard is the recipient of the Vanier Canada Graduate Scholarship. A Kassardjian is the recipient of an Ontario Graduate Scholarship. J-P Julien is supported by operating grant PJT-148811 from the Canadian Institutes of Health Research, the CIFAR Azrieli Global Scholar program, the Ontario Early Researcher Awards program, and the Canada Research Chairs program.