Introduction
In the previous chapter a set of experim ents on DNA im m unisation against hCEA were described. Variable and w eak im m une responses w ere the features of those experiments. The w ork described in this chapter sought to explore the reason(s) for the poor responses described in the light of present understanding of T and B cell activation. According to the current paradigm of T cell activation, both antigen specific and costim ulatory signals are required for activation of naive T cells. The second costim ulatory signals for T cell activation, are provided by ligation of T cell surface molecules such as CD28 or cytokines such as lL-2 that function in an autocrine or paracrine fashion (Jenkins and Johnson 1993). Two types of signals are also required for B cell activation to T -dependent antigens. A m odel of B cell activation is based on positive signals from activated Th cells via CD40 ligation that in tu rn induces Fas expression on B cells and could result in negative signals and apoptosis. The negative signals are overridden by triggering of the m em brane Ig (mlg) w hen native antigen binds to mlg in germinal centres. B cells that have received CD40 and m lg signals would differentiate in response to cytokines such as lL-4, lL-13 and lL-10 (van Kooten and Banchereau 1997). A ctivation of T cells to express CD40 ligand (L) and secrete cytokines for B cell proliferation and differentiation depends on accumulation of positive intracellular signals to a th resh o ld level th ro u g h antigen specific (TCR) an d costim ulatory (CD28) pathw ays (Klaus, Pinchuk et al. 1994). The absence of detectable responses in som e of the anim als im m unised in our studies m ight have been because an activation threshold h ad not been reached. Experim ents described in this chapter w ere designed to augm ent the costim ulatory signals by the use of lipopolysaccharides (LPS) or tetanus toxin fragment-c (Fr-c).
It has been demonstrated that LPS can act as an adjuvant for a variety of different antigens. LPS can cause an increase in antibody responses against
Echis ocellatus venom or sheep red blood cells in mice and tetanus toxoid in rats (Laing and Theakston 1993). This may have been m ediated by TN F-a in the rat system. In a stu d y by Parks et al. it w as show n th at LPS converted the tolerogenic antigen, deaggregated hum an gam m a globulin (DHGG), to an im m unogen (Parks, W alker et al. 1981). In this study, cell transfer experim ents show ed that the use of LPS resulted in activation of antigen specific Th and B cells. In vitro studies have dem onstrated that LPS activated B cells can act as antigen presenting cells. In one study it was show n that LPS activated B cells can act as accessory cells for a con-A response or present chicken ovalbum in to Th cells (Krieger, Gramm er et al. 1985). In another study LPS activated B cells in duced CTL responses against vesicular stom atitis v iru s (VSV) in vitro (Ciavarra and Burgess 1988). Therefore, LPS enhances im m une responses and this could be by increasing the second activation signal either by enhancing the antigen presentation ability of B cells or by other m eans such as stim ulating the secretion of cytokines. Hypothetically also in the presence of antigen, antigen specific B cells m ay become preferentially selected to expand d u rin g LPS- induced polyclonal B cell activation.
As m entioned before, one possible explanation for the w eakness of im m une responses to hCLA may have been the lack of T cell help. Therefore we set out to investigate w hether Fr-c could alter the im m unogenicity of hCLA. Tetanus toxin (TT) has been used as a carrier for a synthetic peptide epitope of
Plasmodium falciparum circumsporozoite (CS) protein (H errington, C lyde et al.
1987). Various studies have shown that TT contains T helper epitopes that can be recognised by hum an T cell clones of varied specificity a n d MHC II restriction (Panina Bordignon, Tan et al. 1989; Reece, G eysen et al. 1993). Conjugation of a "universal" T cell determ inant from TT w as show n to result in induction of antibody responses to a Plasmodium falciparum peptide epitope that w as not otherw ise im m unogenic in six strains of mice w ith different H-2 haplotypes (Kumar, Arora et al. 1992). In another study tw o other TT epitopes w ere conjugated to a Plasmodium falciparum epitope from CS protein an d were
show n to result in augm entation of antibody responses against the protein (Valmori, Pessi et ah 1992). The non-toxic fragment-c of TT that encom passes som e of the T helper cell determ inants of TT has been studied as a vaccine against TT. It has been dem onstrated that im m unisation w ith a DNA construct containing the gene for Fr-c resulted in high anti-Fr-c antibody titres and protection against lethal challenge w ith TT (Anderson, Gao et ah 1997). Finally, in a DNA vaccination study, Spellerberg et ah show ed augm entation of anti-Id antibodies against a single chain Fv (scFv) as a result of fusion of Fr-c w ith the scFv (Spellerberg, Z hu et ah 1997). On the basis of these observations w e chose Fr-c to increase imm unogenicity of hCEA and m ade an im m unisation construct that contained hCEADo followed by the gene for Fr-c.
We investigated hum oral and cellular im m une responses in all the im m unisations described in this chapter. Therefore m agnitude, frequency and kinetics of antibody responses to hCEA and in the case of the Fr-c experim ents to both hCEA and Fr-c were studied. As in our previous experience Th recall responses to hCEA could be dem onstrated, how ever, antibody isotypes were determ ined as a m eans of investigating the type of the im m une response induced by different im m unisation constructs. Two types of im m une responses are thought to be m ediated by T hl and Th2 cell subsets. In mice T h l cells predom inantly secret IL-2 and IFN-y and are associated w ith IgC2a secretion, whereas, Th2 cells secrete IL-4 and IL-5 and are associated w ith production of IgG l (Mosmann, 1986; Stevens, 1988). Therefore, in order to determ ine w hat type of Th cell m ay be involved in an im m une response, the T cells are restim ulated in vitro and the profile of the cytokines secreted is determ ined by ELISA or ELI-spot assays. The cytokine profiles of Th cells could not be determ ined, since in vitro restim ulation of Th responses after DNA vaccination proved to be difficult. Alternatively, determ ination of the antibody isotypes could indirectly point tow ards the Th type involved in the im m une response. In some strains of mice such as C57BL/6 that contain the Ighl-b allele, the gene for IgG2a is deleted (Martin, Brady et ah 1998). These mice express a different
isotype called IgG2c. In our experim ents, for isotype detection w e u sed a commercial antibody that was raised against IgG2c (see m aterial and m ethods). However, it is not know n whether isotype switching to IgG2c occurs as a result of IFN-y signalling or any other events that occur as p art of a T h l response. In our experim ents the mice produced either predom inantly IgG l or IgG2c w ith different treatments. This could be reasonably interpreted as tw o different types of response b u t cannot be categorised definitively as T hl and Th2-
Finally, in this chapter the necessity of fusion of the antigen to the im m unom odulatory Fr-c was studied by com paring the im m unisation of the fusion construct to co-immunisation of separate hCEADo and Fr-c constructs. In addition, an experiment was perform ed that dem onstrates prim ing of CTL in response to DNA im m unisation and provides su p p o rt for the hypothesis th at the failure of CTL detection after our DNA im m unisations m ay be due to a low frequency of CTL precursors.
M ethods
V a c c in a tio n c o n s tr u c ts
For the experim ents on the adjuvant effect of LPS, p V A C /em p ty and pV A C /hC E A that w ere described in chapter 3 w ere used. pcD N A 3/em pty, pcD N A S/hC EA D o and pcDNA3/hCEADoFr-c w ere used in the rem aining experiments of this chapter. hCEADo fragm ent w as cut out of pV A C/hCEAD o using Hind III and Xba I restriction sites and inserted into the same sites of the pcDNA3 vector. hCEADoFr-c fusion w as achieved in tw o stages. First, hCEADo and Fr-c w ere separately amplified. The 3' prim er for hCEADo and the 5' prim er for Fr-c had complementary sequences that extended beyond the sequences of their corresponding target genes. The com plem entary overlapping nucleotides w ere designed to code for a linker p ep tid e of Gly-Pro-Gly-Pro sequence. In a second round of amplification the two PCR products w ere mixed after gel purification and a set of primers were used that bound to the 5' end of hCEADo and the 3' end of Fr-c genes. This fusion gene was cut by H ind 111 and N ot 1 restriction enzymes, gel purified and inserted into the corresponding sites of pcDNA3. hCEADo and hCEADoFr-c w ere sequenced in o rd er to detect possible m utations introduced by the PCR procedure, however, such m utations w ere not detected. Expression of protein by pcDNA3 vectors was not tested in vitro, how ever, in the experim ents described in this chapter it w ill become apparent that all these constructs, regardless of the presence of Fr-c, induced antibody responses specifically against hCEA. This indicates th at the roteins coded by these vectors were expressed, in vivo.
I m m u n is a ti o n sch edu le
Mice were im m unised in the quadriceps muscles u nder anaesthesia. For the experiments on the adjuvant effect of LPS, control mice were given a single dose of 100 pg DNA at 1 m g /m l in saline. The experim ental gro u p s w ere injected w ith the same dose of DNA, which had been spiked w ith 5 or 10 EU of LPS. The batch of DNA used in this experim ent w as p re p a re d u sin g a commercial kit that incorporated an endotoxin removal step (see m aterial and methods). For the experiments on the adjuvant effect of tetanus toxoid fragm ent c (Fr-c) mice were imm unised with 50 pg and boosted twice w ith 50 pg of DNA at three w eeks intervals. This protocol w as used by Spellerberg et al. for im m unisation w ith a scFv-Fr-c fusion construct (Spellerberg, Zhu et al. 1997).
M e a s u r e m e n t o f a n t i b o d y le vels
An Enzyme Linked Im munosorbant Assay (ELISA) was used to m easure anti-hCEA or anti-Fr-c antibody levels. The ELISA plates w ere coated w ith hCEA or Fr-c protein in PBS. The mAh units w ere based on the A5B7 mAh. O D450 curves for different dilutions of the standard and the sam ples w ere constructed. The A5B7 standard curves w ere constructed such that the optical density obtained w ith Im g /m l of A5B7 in each ELISA plate w as taken to be equivalent to 1000 mAb units (see the m ethods chapter). An OD450 reading of a sam ple dilution that was in the linear p a rt of the dilution curve w as used to read the mAh units for that OD450 in the standard curve and then m ultiplied by the sample dilution to obtain the mAh units of anti-hCEA antibody in the s e ru m .
The antibody isotypes w ere determ ined as above except th at HRP- conjugated anti-m ouse isotype specific instead of anti-m ouse polyclonal Ig antibodies were used for detection. In C57BL/6 mice the IgG2a gene is deleted (Martin, Brady et al. 1998). In our experiments, a set of comm ercially p rep ared
antibodies th at w ere specifically developed against antibodies of various isotypes of C57BL/6 mice origin, were used.
All the anti-Frc ELISA assays w ere perform ed by Dr. C.A. King at the Molecular Immunology Group, Southampton University Hospital, UK.
R esults
LP S a s an a d j u v a n t f o r D N A v a c c in a tio n
This experim ent was perform ed to assess w hether LPS can act as an adjuvant. The plasm id DNA used in this experim ent w as prep ared using a commercial kit that had an LPS extraction step. After LPS extraction, different am ounts of LPS were added in a fixed volume to aliquots of the same batch of DNA. Three groups of eight mice were injected in the quadriceps muscles w ith 100 pg of pVAC/hCEA mixed w ith 0, 5 or 10 endotoxin units (EU) of LPS and boosted 2 w eeks later w ith 50 pg of the sam e p rep aratio n . For control pV A C /em pty mixed with varying levels of LPS w as injected in the same way, in to groups of 4 or 6 mice.
At four weeks post im m unisation there w ere not any detectable anti hCEA antibody responses. Figure 4.1 shows a low level of anti hCEA response in all the g ro u p s im m unised w ith p V A C /h C E A at six w eeks p o st im m unisation. Positive responses were considered to be the ones th at w ere m ore than two standard deviations above the controls. There w ere 2, 5 and 2 responders in the group that received 0, 5 or 10 EU of LPS, respectively. One of the mice that were injected with 10 EU of LPS died after the first injection.
3 0 0 0 ' Hi CO < e 2 0 0 0' 1 0 0 0' A n ti-h C E A a n t ib o d y r e s p o n s e w i t h in c r e a s in g l e v e l s o f L P S a s a n a d j u v a n t CL g CL P tu + Cl