B cell targeting by molecular adjuvants for enhanced immunogenicity

Figures & data

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

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

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