This screen is designed to identify genes required for the in vivo B cell response to a model antigen encoded by a recombinant Semliki Forest Virus vector. Mice are immunized on day 0, boosted on day 14, and serum is collected and analyzed for antigen-specific IgG on day 28. ENU-mutatgenized G3 mice that produce a reduced amount of IgG to a strong antigen or a detectable amount of IgG to a weak antigen (see below) relative to wild type mice are identified as potential mutants.

 

During a T cell-dependent humoral immune response, a subset of activated CD4⁺ cells -- follicular homing T cells or T-FH -- migrate to the T-B borders of secondary lymphoid organs, and interact with cognate antigen-specific conventional (B-2) B cells that have been activated by soluble antigen, and stimulate them through CD40-CD40 ligand (1) and other interactions to expand within T cell or B cell zones. B cells in the T cell zone develop into short-lived plasma cells that produce germline-encoded antigen-specific antibody. In contrast, B cells expanding in B cell zones generate regions called secondary follicles, precursors of the germinal center (GC) reaction. As the GC reaction progresses, it becomes polarized into separate “dark” and “light” zones. B cells proliferate and diversify via somatic hypermutation within the dark zone, and then migrate to the light zone, where B cells with high-affinity variant Ig receptors are selected to either re-enter the GC cycle or exit the GC reaction. Cells exit the GC reaction as either long-lived plasma cells or recirculating memory B cells. Long-lived plasma cells produce large amounts class-switched, high-affinity antibody. Memory B cells do not actively secrete antibody. Instead, these cells are programmed for expansion and differentiation into high-affinity plasma cells upon secondary encounter with antigen, the hallmark of the memory B cell response.

 

Single-round infectious recombinant viral vectors, including recombinant Semliki Forest Virus (rSFV) vaccine vectors (2-5), induce both cellular and humoral immune responses. SFV is an alphavirus genetically related to Sindbis virus and Venezuelan equine encephalitis virus, for which single-round replicon systems have also been developed (6-8). The vector used here contains the SFV translation-enhancer element upstream and in frame with an internal signal sequence and the target antigen-encoding sequence (9). This vector was shown to result in secretion of ten-fold more antigen compared to the same vector lacking the enhancer element and using the N-terminal CD5 signal sequence, as well as improved antibody titres in the sera of immunized mice (9). In the protocol outlined here, the rSFV vector encodes either the model antigen β-galactosidase (GAL) or ovalbumin (OVA) and is administered via intraperitoneal injection. Detection of GAL- or OVA-specific IgG two weeks after administration of a second dose of the vector is used as a readout for the class-swiched B cell response.  Most wild type mice mount a robust GAL-specific IgG response, and mutagenized mice that fail to do so may possess mutations in genes required for B or CD4⁺ T cell development or activation, isotype class switching, or terminal differentiation.  By contrast, wild type mice fail to produce detectable OVA-specific IgG responses; of the strains tested, only interferon (IFN)α receptor-deficient mice mount an OVA-specific IgG response.  Mutagenized mice that are able to respond may have mutations in the type I IFN signaling pathway or other genes that normally downregulate weak B cell responses.

 

β-galactosidase (Roche Applied Science, Indianapolis, IN)

 

ovalbumin (Sigma-Aldrich, St. Louis, MO)

 

HRP-conjugated goat-anti-mouse IgG (Southern Biotech, Birmingham, AL)

 

TMB SureBlue reagent (KPL, Inc., Gaithersburg, MD)

 

TMB Stop Solution (KPL, Inc., Gaithersburg, MD)

Priming

1. On day 0, dilute in a total volume of 200 μl 0.9% saline and inject intraperitoneally into each G3 mouse:

a)       2x10⁶ infectious units (IU) of rSFV vector encoding β-galactosidase (GAL), or

b)       10⁷ IU of rSFV vector encoding ovalbumin (OVA).

 

Boosting (Same procedure as priming)

2. On day 14 after priming, dilute in a total volume of 200 μl 0.9% saline and inject intraperitoneally into each G3 mouse:

a)       2x10⁶ infectious units (IU) of rSFV vector encoding β-galactosidase (GAL), or

b)       10⁷ IU of rSFV vector encoding ovalbumin (OVA).

 

Measurement of antigen-specific IgG by ELISA

3. Prepare an ELISA plate. Coat a 96-well round bottom plate overnight at 4°C or for 2 hours at room temperature with:

a)       2 μg/mL β-galactosidase in PBS, or

b)       10 μg/mL ovalbumin in PBS.

 

4. On day 28 after priming, collect about 50 μl of blood from the orbital sinus of each mouse. For the detection of GAL-specific IgG, dilute 2 μL of sera in 1 mL cold 1% (w/v) milk in PBS (prepare serum dilutions in eppendorf tubes). For the detection of OVA-specific IgG, dilute 5 μL of serum in 250 μL of cold 1% (w/v) milk in PBS (prepare serum dilutions in separate eppendorf tubes).

 

5. Perform ELISA according to standard protocol:

a)       Snap plate to remove the coating antigen.

b)       Wash plate 3 times with 200 μL of PBS per well.

c)       Add 200 μL of 5% (w/v) milk in PBS per well.

d)       Wrap or cover plate and incubate at least 1 hour and up to 3 hours at 37°C.

e)       Wash plate 3 times with 200 μL of PBS per well.

f)        Add 100 μL of 1% (w/v) milk in PBS per well.

g)       Add 100 μL of diluted serum to appropriate well containing 100 uL of 1% (w/v) milk.  

h)       Make at least 4 serial dilutions at 1:2 from this well, discarding the last 100 μL.

i)        Wrap or cover plate and incubate for exactly 1 hour at 37°C.

j)        Wash plate 6 times with PBS + 0.05% Tween-20.

k)       Add 100 μL of 1% milk containing HRP-conjugated goat-anti-mouse IgG diluted 1:5,000 per well.

l)        Wrap or cover plate and incubate for exactly 1 hour at 37°C.

m)      Wash plate 6 times with PBS + 0.05% Tween-20.

n)       After last wash has been removed, develop plate by adding 100 μL of room temperature TMB SureBlue reagent per well.

o)       Incubate for 1 to 5 minutes at room temperature.

p)       Stop the reaction by adding 100 μL of TMB Stop Solution per well.

q)       Read absorbance at 450 nm.

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