DNA Vaccine Molecular Adjuvants SP-D-BAFF and SP-D-APRIL Enhance Anti-gp120 Immune Response and Increase HIV-1 Neutralizing Antibody Titers
Product# 1103 HIV-1 p24 Monoclonal Antibody
It is generally believed that broadly neutralizing antibodies (bNAbs) are essential for the prevention of HIV-1 infection. Although several bNAbs have been isolated from HIV-infected individuals (1,–4), they are not commonly generated in most humans following HIV-1 infection. Not surprisingly, a vaccine design that can induce high-titer bNAbs and immunological memory remains a major challenge. HIV-1 has unique structural features, including high variability of protein sequences, inaccessibility of the conserved structures in the Env protein to bNAbs, and extensive glycosylation masking Env antigens (Ags). Further compounding the issue is the potential loss of Env-specific B cell clones that are autoreactive and are therefore deleted during the process of immunological self-tolerance (5,–7). Although most B cells are variably autoreactive in both humans and mice, the majority of B cells join the immunocompetent mature B cell repertoire (8). This suggests that only B cell clones with autoreactivity beyond a certain threshold are deleted by negative selection, while the rest continue their development into mature B cells. Thus, the mature B cell repertoire likely contains weakly HIV-1 reactive B cell clones, as well as some strongly reactive B cell clones that escape tolerance checkpoints or assume an anergic state. Recent evidence suggests that the mature B cell repertoire in both mice and humans contains clones that bind HIV-1 Env and are often polyreactive (9, 10). Thus, in principle, it should be possible to design vaccines targeting HIV envelope-specific B cells that can serve as precursors for anti-HIV-1 bNAbs (7, 11).
A successful HIV vaccine design not only has to overcome some unusual evasive properties of HIV-1 but also has to mimic native Env trimers, which are highly unstable (12). In addition, a vaccine should promote sufficiently high titers of bNAbs that are not diluted by immune responses to nonprotective epitopes. Most antibody responses depend on peptide-antigen presentation by dendritic cells (DCs) to CD4 T cells, upregulating the CD40 ligand (CD40L), which interacts with CD40 on B cells, providing cognate help to B cells. B cells stimulated in this manner migrate to the germinal centers (GCs), where they undergo isotype class switch recombination (CSR) and affinity maturation and differentiate into antibody-secreting plasma cells (ASCs). However, vaccines that mobilize this pathway exploit limited neutralizing epitopes on HIV-1 Env. HIV antigens can also activate B cells directly. Direct engagement of B cells with antigen may increase the number and complexity of the antibody-inducing epitopes available, including glycan-linked peptides. In this mode of immune activation, B cells can present antigen and activate the conventional CD4 T helper pathway as well as T cell-independent mechanisms of antibody production. We and others have shown that T cell-independent mechanisms depend heavily on B cell-activating factor (BAFF) receptor and ligand (13,–17).
The BAFF receptors, BAFF-R (BAFF receptor), TACI (transmembrane activator and calcium-modulating cyclophilin ligand interactor), and BCMA (B cell maturation antigen), play crucial roles in many key aspects of B cell biology, including selection and survival during peripheral B cell maturation and the survival and differentiation of antigen-activated B cells and ASCs (18,–21). Both BAFF and APRIL (a proliferation-inducing ligand) can stimulate B cells via BCMA and TACI. However, BAFF activation of TACI requires aggregates of BAFF (22,–25). Additionally, only BAFF binds BAFF-R. Peripheral, transitional, and mature B cells express BAFF-R on their surfaces, whereas TACI is detectable on all peripheral B cells. BCMA is found primarily in plasma cells. Significantly, the expression of both BAFF-R and TACI on the cell surface is rapidly induced upon the engagement of B cell receptor (BCR) with antigen. Heightened expression of TACI and BAFF-R sensitizes antigen-specific B cells to the growth and differentiation signals of BAFF and APRIL (16) during an immune response, including the increased life span of GCs, class switching to IgG, and affinity maturation (26, 27). In fact, BAFF−/− transgenic mice display spontaneous formation of GCs, promoting a Th1-skewed response with IgG2a and IgG3 production (28). Likewise, high serum BAFF concentrations support the survival and class switching of autoreactive B cells (28). In contrast, gene deletion or neutralization of BAFF causes early dissolution of GCs, whereas transgenic overexpression of BAFF results in spontaneous formation of GCs. Further, an antibacterial vaccine formulation expressing BAFF from a viral vector enhanced and extended a protective high-titer antibody response against Pseudomonas aeruginosa (29). Like BAFF, APRIL has been shown to induce T cell-independent isotype class switching, affinity maturation, and antibody production (13, 24, 26, 30). It was thought that BCMA was primarily responsible for the survival of ASCs. However, recent evidence suggests that TACI and BAFF-R are also required (31, 32). Thus, BAFF and APRIL may promote various aspects of class-switched and high-affinity antibody production.
As with other tumor necrosis factor (TNF) family members, stimulation through BAFF-R, TACI, and BCMA depends on the trimerization of BAFF and APRIL or on interactions between B cells and BAFF/APRIL-expressing cells such as B and T cells, monocytes, neutrophils, and macrophages/myeloid cells (24). A prior study using BAFF and APRIL trimers as adjuvants in rabbits showed improved antibody response; however, only tier 1 viruses could be neutralized (33). These observations led us to hypothesize that soluble multitrimers of BAFF and APRIL, as opposed to single trimers, would induce optimal immune activation of B cells and enhance the overall immune response. We have shown previously that TNF superfamily ligands, including BAFF, APRIL, and CD40L, can be formed into soluble multitrimers by using the lung protein SP-D (surfactant protein D) as a scaffold (34,–36). In these studies, DNA vaccination with an SP-D-BAFF adjuvant enhanced cellular immune responses against an HIV-1 Gag antigen. However, we failed to observe a significant increase in antigen-specific antibody titers (34), possibly due to the requirement for additional costimulatory signals in the vaccine formulation.
In the present study, we evaluated multitrimers of BAFF and APRIL as molecular adjuvants together with interleukin-12 p70 (IL-12p70) in an HIV-1 Env gp140 DNA vaccine model. Using this vaccine design, we tested both DNA vaccination and a DNA prime–protein boost vaccination strategy using a recombinant gp120 protein boost. B cell and antibody responses were evaluated following vaccination. Our data show that soluble multitrimeric forms of BAFF or APRIL, in combination with IL-12p70 and membrane-bound Env protein gp140, enhanced the gp120-specific antibody response and titers of tier 1 and autologous vaccine strain tier 2 neutralizing antibodies.
MATERIALS AND METHODS
Construction and preparation of DNA plasmids.
The HIV-1 Env DNA vaccine plasmid (pgp140; clade C, strain 96ZM651), was obtained from the NIH AIDS Reagent Repository program (catalog no. 8660). The secreted HIV-1 Gag DNA plasmid (pGag; also called pScGag) has been described previously (37) and was provided by George N. Pavlakis. Plasmids coding for the 4-trimer soluble forms of murine SP-D-BAFF and SP-D-APRIL were generated in vectors pVAX1 and pcDNA3.1, respectively, as described previously (34). Constructs were cloned such that mouse SP-D from the N terminus to amino acid sequence ALFPDG was fused directly to the mouse TNF superfamily ligand (TNFSFL) extracellular domain, starting from the N-terminal amino acid sequences QLAALQ (BAFF) and MPASSP (APRIL). Plasmid pIL-12p70, encoding mouse single-chain IL-12, was purchased from InvivoGen Inc. All plasmids were propagated in Escherichia coli strain TOP10. Endotoxin-free DNA plasmid preparations were prepared using an EndoFree Giga plasmid kit (Qiagen). Plasmids were then further purified to remove residual endotoxins with additional Triton X-114 extractions as described previously (38). Plasmid endotoxin levels were <0.2 endotoxin unit (EU)/ml for all constructs as confirmed by a Limulus amebocyte lysate (LAL) endotoxin assay (Lonza Inc.).
Preparation of Env protein.
For the preparation of monomeric Env protein, plasmid pTpa-gp120-His was constructed by fusing HIV-1 Env gp120 (clade C, strain 96ZM651) to the first 21 amino acids of human tissue plasminogen activator (t-PA) at the N terminus and to a His tag at the C terminus. For the preparation of Env protein, 293T cells were transiently transfected with the pTpa-gp120-His plasmid by using the GenJet Plus transfection reagent (SignaGen Laboratories) according to the manufacturer's instructions. Supernatants were harvested 48 h after transfection and were purified on a Ni-nitrilotriacetic acid (NTA) affinity column using standard procedures. Briefly, after the separation of cell debris by high-speed centrifugation, the cell culture supernatant was loaded onto a Ni-NTA column, and the column was washed extensively with 20 mM imidazole to remove contaminating proteins. The Env protein was eluted with 200 mM imidazole and was dialyzed to remove excess imidazole. The purified Env protein was then concentrated using an Amicon Ultra-15 centrifugal filter unit with a 100,000-molecular-weight cutoff filter (Millipore Inc.) according to the manufacturer's instructions. The protein concentration was determined by a fluorescence-based Quant-iT assay (Invitrogen) and by spectrophotometry using an extinction coefficient of 37,930/M/cm, calculated using ProtParam online software at the ExPASy server (Invitrogen) (39).
Mice and immunization schedule.
Female BALB/c mice (7 to 8 weeks old) were used in all vaccination experiments. Animals were housed at the University of Miami under the guidelines of the National Institutes of Health (NIH; Bethesda, MD). All animal experiments were performed in accordance with national and institutional guidance for animal care and were approved by the IACUC of the University of Miami. The pGag and pgp140 plasmids were diluted in phosphate-buffered saline (PBS) and were combined with either pcDNA3.1 or one of the SP-D-TNFSFL adjuvant plasmids as noted, with or without pIL-12. Plasmid mixtures were injected intramuscularly (i.m.) into the quadriceps muscles of both hind limbs. DNA vaccinations were given three times at 2-week intervals with 80 μg of the 96ZM651 membrane-bound gp140 plasmid mixed with 20 μg of the pcDNA3.1 or SP-D-TNFSFL adjuvant plasmid, with or without 20 μg of pIL-12. Doses were administered in a total volume of 100 μl PBS (50 μl per limb). For DNA vaccine studies, 2 weeks after the final DNA immunization, mice were anesthetized with ketamine-xylazine, and blood was drawn by cardiac puncture to obtain serum. For heterologous DNA prime–protein boost vaccinations, mice were injected intramuscularly with DNA three times at 2-week intervals, followed 3 weeks later by a single Env protein boost, given by intramuscular injection at the same site. Thirty micrograms of gp120 Env protein was formulated with an equal volume of Freund's incomplete adjuvant in a total volume of 100 μl. The vaccine was then injected intramuscularly at a 50-μl volume into each hind-leg quadriceps muscle. Two weeks following the Env protein boost, mice were anesthetized with ketamine-xylazine, and blood was drawn by cardiac puncture to obtain serum. Following euthanization, spleens were removed for B cell analysis. Half the spleen was quick-frozen in OCT compound for histology. Single-cell splenocyte preparations were obtained for the other half of each spleen by passage through a 40-μm nylon cell strainer (BD Falcon). Erythrocytes were depleted with lysis buffer (Sigma), and splenocytes were washed thoroughly with complete R10 medium (RPMI 1640 medium supplemented with 10% fetal bovine serum [FBS], 50 μM 2-mercaptomethanol, 100 U/ml of penicillin, 100 μg/ml streptomycin, and 10 mM HEPES).
ELISA for anti-Env IgG responses.
Antibody production was measured by an enzyme-linked immunosorbent assay (ELISA). Ninety-six-well ELISA plates were coated with HIV-1 IIIB p55 Gag protein (10 μg/ml; provided by the NIH AIDS Reagent Program) or HIV-1 Env 96ZM651 gp120 protein (10 μg/ml; provided by the NIH AIDS Reagent Program or prepared in the lab) overnight at 4°C. Mouse sera at varying dilutions were added to antigen-coated wells and were incubated at room temperature for 2 h with shaking. After plates were washed, antigen-specific IgG antibodies were detected using horseradish peroxidase-conjugated goat anti-mouse IgA, IgG, IgG1, IgG2b, or IgG2a (Jackson ImmunoResearch Inc.). The signal was developed using the SureBlue TMB microwell peroxidase substrate (Kirkegaard & Perry Laboratories, Inc.). Plates were analyzed using a 96-well plate absorbance reader at 650 nm. Endpoint titers were calculated as the highest dilutions with more than twice the background absorbance of control wells. For the quantification of total IgG, a standard curve using known amounts of purified mouse IgG was used.
HIV-1 neutralization assay.
Virus neutralization was measured using a luciferase-based assay on TZM.bl cells as described previously (40). The TZM.bl cell assay was performed with HIV-1 tier 1 pseudovirus MW965.26 and tier 2 pseudovirus 96ZM651 (vaccine strain). The assay measures the reduction in luciferase reporter gene expression by TZM.bl cells following a 48-h incubation period with a single round of virus infection. The 50% inhibitory dose (ID50) was calculated as the serum dilution that caused a 50% reduction in relative luminescence units (RLU) from the level in the virus control wells after the subtraction of the RLU in cell control wells. Calculations were performed using a validated macro employing a point-based algorithm with linear interpolation between the two replicates on either side of 50% RLU reduction. The level of nonspecific neutralization for each mouse was determined by measuring the neutralization of murine leukemia virus (MLV) with SVA elements (SVA-MLV). Neutralization titers for each mouse were scored as positive only if they were >3-fold higher than SVA-MLV titers.
The avidity of antibodies against gp120 protein was determined by a particle disruption ELISA using the chaotropic agent sodium thiocyanate (NaSCN), as described previously (41). The binding of gp120 protein and incubation with mouse serum were performed as described above. Plates were washed three times with 0.05% PBS–Tween 20, and the chaotropic agent NaSCN, diluted in PBS, was added to replicate wells (0, 1, 2, 3, 4, or 5 M). Plates were first incubated at room temperature for 30 min and then washed six times with PBS–Tween 20. Subsequent ELISA steps were performed as detailed above. The avidity index for each mouse sample was recorded as the mean molarity of NaSCN that reduced the optical density at 650 nm (OD650) by 50% from that for wells treated with PBS.
Spleens from immunized mice were cut transversely; the anterior half was used for flow cytometry and an enzyme-linked immunosorbent spot (ELISPOT) assay, and the posterior half was used for tissue immunofluorescence (see below). Flow cytometry (with an LSR II system; BD Biosciences) was performed to determine the percentages of live splenic and GC B cells, the CD4/CD8 T cell ratios, and the level of purity of the B cell enrichment. Single-cell suspensions of splenocytes were stained with fluorescein isothiocyanate (FITC)-conjugated anti-B220 (RA3-6B2; eBioscience), phycoerythrin (PE)-Cy7-conjugated anti-CD19 (1D3; BD Biosciences), allophycocyanin (APC)-conjugated anti-CD4 (RM4-5; BD Biosciences), Pacific Blue (PB)-conjugated anti-CD8 (53-6.7; BD Biosciences), APC-conjugated anti-CD138 (anti-syndecan-1; BD Biosciences), and biotin-conjugated anti-GL7 (eBiosciences) and V500-conjugated streptavidin (BD Biosciences). Live/dead analysis was performed using LIVE/DEAD near-IR staining (Invitrogen). The values thus obtained were then used to calculate the numbers of ASCs, B cells, and GC B cells per half-spleen.
ELISPOT assay for anti-gp120 B cells.
A single-cell spleen suspension was made as described above. CD43− (total conventional) B cells were enriched using the BD IMag B cell enrichment kit (BD Biosciences). A dilution series of B cells (1 × 106 to 0.5 × 105) was plated in duplicate wells of a Millipore ELISPOT plate with a polyvinylidene difluoride (PVDF) membrane that had been coated 24 h previously with 5 μg/ml gp120 protein. Cells were incubated overnight at 37°C, and then total IgG-producing gp120-specific ASCs were revealed using the mouse IgG ELISpot Plus kit (Mabtech) according to the manufacturer's instructions. The plates were dried and were read on an ImmunoSpot ELISPOT plate reader (Cellular Technology Ltd.). Flow cytometry (on an LSR II system; BD Biosciences) was used to determine the percentage of splenic B cells and the level of purity of the B cell enrichment (with FITC-conjugated anti-B220 [RA3-6B2; eBioscience] and PE-Cy7-conjugated anti-CD19 [1D3; BD Biosciences]). These values were then used to calculate the number of ASCs per spleen.
The remaining half of the spleen (see above) was embedded in OCT compound and frozen in liquid nitrogen, and 5-μm sagittal sections were cut. For staining, slides were incubated for 10 min in −20°C acetone, washed in Tris-buffered saline (TBS), and blocked in 3% bovine serum albumin (BSA) in TBS with 0.1% Tween 20 for 1 h at room temperature. Slides were then incubated with goat anti-mouse CD3 (M20; Santa Cruz Biotechnology), biotin-conjugated anti-CD19 (1D3; BD Biosciences), and tetramethyl rhodamine isocyanate (TRITC)-conjugated peanut agglutinin (PNA) (Sigma) in blocking buffer at 4°C overnight. Slides were first washed with TBS and then incubated for 1 h with an Alexa Fluor 647-conjugated anti-goat antibody (Life Technologies) and Alexa Fluor 488-conjugated streptavidin. Slides were washed with TBS and were then mounted using ProLong Gold Antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI). Entire slides were digitally scanned using the VS120 virtual slide scanning system (Olympus America, Inc.) at ×40 magnification. The B cell areas in germinal centers were measured using ImageJ.
GraphPad Prism software, version 6.0, was used to calculate significance by one-way analysis of variance (ANOVA) with multiple comparisons using Tukey's post hoc analysis. Correlation analysis was performed by linear regression using GraphPad Prism software, version 6.0.
Combining DNA vaccine adjuvant pSP-D-BAFF with pIL-12 increased antibody titers.
Previous studies with DNA vaccines containing the pSP-D-BAFF adjuvant plasmid have shown only low levels of antibody titers relative to those with an antigen-only control vaccine (34). To explore methods of enhancing antibody titers from DNA vaccines, we performed a DNA vaccination experiment on mice with HIV-1 Gag DNA (as a model antigen) combined with plasmid pSP-D-BAFF or pIL-12, a single-chain IL-12p70 construct expressing both IL-12p35 and IL-12p40 joined by a flexible linker (42). As shown in Fig. 1B, pSP-D-BAFF failed to increase anti-Gag antibody titers, while the pIL-12 adjuvant significantly increased antibody titers as measured by ELISA. Next, we moved to an HIV-1 gp140 DNA vaccine model to test the ability of combinations of IL-12p70 and SP-D-BAFF adjuvants to enhance antibody titers. Plasmid pgp140 (encoding membrane-bound gp140 of HIV-1 clade C strain 96ZM651) was combined with either the empty vector pcDNA3.1, pIL-12 plus pcDNA3.1, or pIL-12 plus pSP-D-BAFF. Again, addition of the IL-12p70 adjuvant significantly increased mean antibody titers over those with pgp140 alone (Fig. 1C). Mean antibody titers were also increased with the combination of pgp140, pIL-12, and pSP-D-BAFF over those with gp140 antigen alone. Although we observed a trend toward higher antibody titers with the combination of the pIL-12 and pSP-D-BAFF adjuvants than with pIL-12 alone, the difference was not statistically significant, a result consistent with those of previous DNA vaccination studies (34, 35).
IL-12p70 is necessary for the induction of antibodies by DNA vaccines encoding SP-D-BAFF. Mice were vaccinated i.m. with 80 μg of an antigen plasmid mixed with 20 μg of either an empty vector, the IL-12p70 single-chain dimer, SP-D-BAFF DNA, or a combination of the latter two (120 μg total DNA per mouse). (A) Animals were vaccinated every 2 weeks for a total of 3 vaccinations. Serum was obtained 2 weeks following the final vaccination and was assayed for binding to the antigen by ELISA. (B) The adjuvant plasmid pIL-12 induced higher antibody titers than did Gag alone or Gag plus SP-D-BAFF. (C) When HIV-1 clade C 96ZM651 gp140 was used as the antigen, either pIL-12 alone or pIL-12 plus pSP-D-BAFF induced higher titers of antibody than the antigen alone.
HIV-1 gp140 DNA vaccines expressing IL-12p70 plus SP-D-BAFF increased tier 1 and tier 2 neutralization antibody titers.
We next assessed the antibody titers and neutralization activity of vaccines incorporating SP-D-BAFF or the related construct SP-D-APRIL. Experiments again used an HIV-1 gp140 DNA vaccine mouse model. Vaccine groups included pgp140 HIV-1 Env antigen and the adjuvant plasmid pIL-12. As shown in Fig. 2B, all vaccine groups induced anti-Env antibody responses as measured by IgG ELISAs. The mean antibody titer with the SP-D-APRIL adjuvant was lower than that with SP-D-BAFF, but the difference did not reach statistical significance. Vaccines induced both IgG2a and IgG1 antibodies, suggesting a balanced Th1 and Th2 response. Next, sera from vaccinated mice were assayed for HIV-1 neutralization by a TZM.bl assay. As shown in Fig. 2C, pIL-12 plus pSP-D-BAFF significantly enhanced serum neutralization titers over those with the pIL-12 adjuvant alone. Vaccination with the BAFF adjuvant enhanced the neutralization of both tier 1 (MW965.26) and tier 2 (vaccine strain 96ZM651) viruses. Mice vaccinated with both the pIL-12 and pSP-D-APRIL adjuvants showed higher mean neutralization titers against tier 1 virus than those given the pIL-12 adjuvant alone, but the difference did not reach statistical significance (P = 0.076).
HIV-1 neutralization correlated with IgG2a antibody titers. Neutralization titers (both tier 1 and tier 2) were correlated with anti-gp120 IgG2a (A) or IgG (B) ELISA values (OD650) at a 1:480 serum dilution. Mice from all vaccine groups with a measurable neutralization titer were included in the analysis.
Evaluation of SP-D-BAFF and SP-D-APRIL by DNA prime–protein boost vaccination.
DNA prime–protein boost vaccination is a promising approach for the generation of anti-HIV-1 Env neutralizing antibodies (43, 44). To extend our encouraging results with SP-D-BAFF and SP-D-APRIL, we investigated whether the performance of these adjuvants could be further enhanced by a protein boost. Vaccination was performed with three biweekly DNA prime injections, followed by a single protein boost 3 weeks after the final DNA vaccination (Fig. 4A). Antibody responses were measured 2 weeks following the protein boost. All groups generated high titers of anti-gp120 IgG antibody following the protein boost (Fig. 4B). No significant differences in IgG, IgG1, IgG2b, or IgA titers were observed between groups. However, the differences in IgG2b titers between the groups given SP-D-BAFF or SP-D-APRIL and the group receiving gp140 plus IL-12p70 did approach significance (P = 0.0744). Importantly, IgG2a antibody titers showed a significant difference at a 10−5 serum dilution between groups given SP-D-BAFF or SP-D-APRIL and groups vaccinated with gp140 plus IL-12p70 alone. Endpoint titers were also determined. As shown in Fig. 4D, there was no significant difference in endpoint titers between groups.
SP-D-APRIL significantly increased vaccine strain tier 2 neutralization titers in a DNA prime–protein boost vaccine model. The same sera used for the experiment for which results are shown in Fig. 4 were assayed for neutralization of tier 1 (MW965.26) or tier 2 (vaccine strain 96ZM651) HIV-1. (A) Tier 1 neutralization activity. Protein boosting gave similar tier 1 neutralization titers for all groups. (B) Tier 2 neutralization titers were significantly enhanced with the addition of the SP-D-APRIL adjuvant, and the enhancement obtained with SP-D-BAFF approached significance (P = 0.0604). Neutralization titers were considered positive if values were >3 times the titer against a murine leukemia virus control.
SP-D-BAFF and SP-D-APRIL significantly increased antibody avidity and the number of gp120-specific antibody-secreting cells.
BAFF and APRIL are known to enhance the germinal center reaction and the expression of activation-induced cytidine deaminase (AID), an enzyme essential for somatic hypermutation in complementarity-determining region 3 (CDR3). AID activity enhances antigen binding as well as immunoglobulin isotype class switch recombination, which determines the localization and function of the antibody molecule (26, 45,–47). We hypothesized that a possible mechanism of action of the SP-D-BAFF and SP-D-APRIL adjuvants was to increase the germinal center reaction, enhancing the affinity maturation of Env-specific B cells and thereby leading to enhanced neutralization. To assess affinity maturation, sera from vaccinated animals were evaluated for anti-gp120 functional avidity by ELISA in the presence of increasing concentrations of sodium thiocyanate (NaSCN). As shown in Fig. 6A, addition of either pSP-D-BAFF or pSP-D-APRIL during the DNA prime significantly increased the IgG avidity index over those for all other groups. In contrast, SP-D-CD40L did not significantly increase avidity.