Memórias do Instituto Oswaldo Cruz Fundação Oswaldo Cruz, Fiocruz ISSN: 1678-8060 EISSN: 1678-8060 Vol. 97, Num. 1, 2002, pp. 95-99

Mem Inst Oswaldo Cruz, Rio de 
  Janeiro, Vol. 97(1) 2002, pp. 95-99
Additives and Protein-DNA Combinations 
  Modulate the Humoral Immune Response Elicited by a Hepatitis C Virus Core-encoding 
  Plasmid in Mice  
Liz Alvarez-Lajonchere/+, Santiago 
  Dueñas-Carrera, Ariel Viña, Thelvia Ramos*, Dagmara Pichardo, 
  Juan Morales  
HCV Department, Vaccine Division, Centro de Ingeniería 
  Genética y Biotecnología *Centro Nacional de Genética Médica, 
  Casilla Postal 6162, Havana City, Cuba 

  +Corresponding author. Fax: +53-7-214764. E-mail:  
  juan.morales@cigb.edu.cu 
Received 20 February 2001 

  Accepted 10 October 2001 
Code Number: oc02017 
Humoral and cellular immune responses are 
  currently induced against hepatitis C virus (HCV) core following vaccination 
  with core-encoding plasmids. However, the anti-core antibody response is frequently 
  weak or transient. In this paper, we evaluated the effect of different additives 
  and DNA-protein combinations on the anti-core antibody response. BALB/c mice 
  were intramuscularly injected with an expression plasmid (pIDKCo), encoding 
  a C-terminal truncated variant of the HCV core protein, alone or combined with 
  CaCl2, PEG 6000, Freund's adjuvant, sonicated calf thymus DNA and 
  a recombinant core protein (Co.120). Mixture of pIDKCo with PEG 6000 and Freund's 
  adjuvant accelerated the development of the anti-core Ab response. Combination 
  with PEG 6000 also induced a bias to IgG2a subclass predominance among anti-core 
  antibodies. The kinetics, IgG2a/IgG1 ratio and epitope specificity of the anti-core 
  antibody response elicited by Co.120 alone or combined with pIDKCo was different 
  regarding that induced by the pIDKCo alone. Our data indicate that the antibody 
  response induced following DNA immunization can be modified by formulation strategies. 
   
Key words: hepatitis C virus - DNA immunization 
  - core  
It is now well established that injection of 
  plasmid DNA through a wide range of routes induces both humoral and cellular 
  immune responses against the encoded proteins in several hosts. Moreover, immune 
  responses induced by DNA immunization have lead to protection against various 
  viral, bacterial and parasitic pathogens (Donnelly et al. 1997). However, immune 
  responses generated by this methodology against several antigens remain insufficient 
  (Hasan et al. 1999). While the majority of the experiments conducted to date 
  have used phosphate buffered saline pH 7.5 (PBS) or saline as a diluent for 
  the injected DNA, a great deal of effort is now being applied to the development 
  of delivery vehicles and adjuvants. Such reagents may increase the uptake of 
  DNA, reduce the necessary dose for immunization and enhance subsequent immune 
  responses. Some systems currently under investigation are cationic liposomes 
  (Gregoriadis et al. 1997), immuno-stimulatory oligonucleotide sequences (Klinman 
  et al. 1997), cytokines (Chow et al. 1997) and monophosphoryl lipid A (Sasaki 
  et al. 1998).  
In this paper, different formulations were investigated 
  to modulate the antibody response generated by a DNA vaccine. We studied the 
  humoral immune response elicited by vaccination with a plasmid encoding a truncated 
  variant of the hepatitis C virus (HCV) core protein. Several B cell and cytotoxic 
  T-lymphocyte determinants within the HCV core protein have been characterized 
  (Walker 1996, Jackson et al. 1997). Moreover, of the various regions of HCV, 
  antibodies against the core protein are often the first to appear during HCV 
  natural infection (Okamoto et al. 1992). However, plasmid expressing the HCV 
  nucleocapsid alone often elicited strong cellular but weak and/or transient 
  humoral immunity (Lagging et al. 1995, Chen et al. 1995). Consequently, the 
  HCV core antigen is a good model for investigations about the modulation of 
  antibody response generated following DNA immunization. We particularly investigated 
  different additives to facilitate the plasmid stability and uptake by the cells. 
  Use of a classical adjuvant like Freund and the combination of plasmid DNA with 
  a recombinant core protein were also evaluated to obtain an improved anti-core 
  antibody response. 
MATERIALS AND METHODS 
  
Co.120 protein - Recombinant Co.120 is 
  an Escherichia coli-derived protein containing the first 120 amino acids 
  of HCV viral polyprotein. It was purified by a combination of washed pellet 
  procedures and gel filtration chromatography as previously described (Dueñas-Carrera 
  et al. 1999). Briefly, recombinant protein was expressed in BL21 (DE3) E. 
  coli cells. For purification, expressing cells were disrupted with French 
  press (Braund, 1,500 kg/cm2) at 1 g/ml in 10 mM Tris HCl, 6 mM EDTA, 
  pH 8. The insoluble fraction of cell lysate was washed with 0.5 M urea, 1% Triton 
  X-100, 10 mM EDTA, 300 mM NaCl, 10 mM 2-mercaptoethanol, 10 mM Tris HCl pH 8 
  buffer. Co.120 protein was solubilized by increasing the concentration of the 
  urea up to 2 M. The supernatant was desalted on a Sephadex-G25 column equilibrated 
  with carbonate buffer, pH 10.6. Desalting by gel filtration rendered soluble 
  Co.120 protein at 95% of purity.  
Human sera - Human sera were obtained 
  from blood donors and chronic patients and previously screened for the presence 
  of anti-HCV antibodies by UMELISA HCV from Centro de Inmunoensayo (La Habana, 
  Cuba). The anti-HCV positive sera were also confirmed by Ortho HCV 2.0 ELISA 
  (Ortho Diagnostic Systems, Raritan, NJ).  
Plasmids - pAEC-K6: expression vector 
  that contains the human cytomegalovirus immediate early promoter, simian virus 
  40 terminator and polyadenylation sequences, a bacterial replication origin 
  from pUC and a kanamycin resistant gene as selection marker. pIDKCo: an expression 
  plasmid generated by inserting a 528 nucleotide DNA fragment, coding for the 
  first 176 aa of the HCV core protein, into the compatible sites of the pAEC-K6 
  (Dueñas-Carrera et al. 2000).  
E. coli strain XL-1 Blue [(F', proAB, 
  lacIqDZM15, Tn10), endA, hsdR17, supE, 
  thi-1, recA1, gyrA96, relA1, lac.] was transformed with pIDKCo and pAEC-K6 plasmids. 
  Cells were grown under selective pressure with 50 mg/l kanamycin. Plasmid DNA 
  was subsequently purified as previously described (Horn et al. 1995).  

Animals and immunization schedule - BALB/c 
  female mice of 6 to 8 weeks old (18-20 g of weight) were purchased from CENPALAB 
  (Ciudad de la Habana, Cuba) and hosted in appropriated animal care facilities 
  during the experimental period. The animals were handed following the international 
  guidelines required for experimentation with animals. Mice were injected at 
  0 and 3 weeks, in the quadriceps muscle, with 100 µl of different immunogens. 
  Blood samples were collected from the retro-orbital sinus at 0, 5, and 14 weeks 
  after the primary immunization. The mice were euthanized after the final blood 
  samples were taken. The study included 10 groups of 5 mice each. The groups 
  were conformed as follows: group 1 (pAEC-K6) was injected with a mock DNA (pAEC-K6 
  plasmid). The second group (Co.120-pIDKCo) received pIDKCo combined with the 
  protein Co.120. Both components were blended and stirred overnight. This mixture 
  was centrifuged for 15 min at 3,000 xg and the supernatant was given as immunogen. 
  The third (Co.120) and fourth (pIDKCo) groups received the Co.120 protein and 
  the pIDKCo alone, respectively. Groups 5 (CaCl2), 6 (PEG 6000), 7 
  (Freund's) and 8 (sCT-DNA) were immunized with pIDKCo combined with 100 mM CaCl2 
  (Merck, Darmstadt, Germany), 1% PEG 6000 (Merck, Darmstadt, Germany), Freund's 
  adjuvant (Sigma, St Louis, USA) and 100 µg of sonicated calf thymus DNA 
  (sCT-DNA) (Promega, Madison, USA), respectively. Group 9 (pIDKCo/Co.120) was 
  primed with pIDKCo and boosted with Co.120. The last group (Co.120/pIDKCo) was 
  primed with Co.120 and boosted with pIDKCo. Plasmids were administered at 1 
  µg/µl and Co.120 protein at 0.1 µg/µl in PBS.  

Enzyme-linked immunosorbent assay (ELISA) 
  - To detect murine anti-core antibodies, 96-well microtiter plates (Costar, 
  Cambridge, MA, USA) were coated with 100 µl of Co.120 (10 µg/ml) 
  diluted in coating buffer (50 mM carbonate buffer, pH 9.6) overnight at 4ºC. 
  The wells were washed three times with 0.05% Tween 20 in PBS (PBST) and blocked 
  with 200 µl of PBST containing 1% skimmed milk (Oxoid, Basingstoke, Hampshire, 
  England) for 1 h at room temperature. After three washes with PBST, 100 µl 
  of serial two-fold dilutions of individual mouse sera in PBST were added and 
  incubated at 37ºC for 1 h. The plates were washed three times with PBST, 
  and 100 µl of horseradish peroxidase-conjugate goat anti-mouse IgG (Amersham, 
  Little Chalfont, Bucks, UK) 1:3000 diluted was added at 37ºC for 1 h. For 
  subtyping of mouse antibodies, 1:50 dilution of pooled sera from each group 
  was used. Biotinylated anti-mouse IgG of the appropriate subclass (Amersham, 
  Little Chalfont, Bucks, UK) was added at 1:1000 dilution, followed by a streptavidin-biotinylated 
  horseradish peroxidase conjugated (Amersham, Little Chalfont, Bucks, UK) diluted 
  1:3000 and incubated for 30 min at 37ºC. Positive reactions were visualized 
  with o-phenylenediamine (Sigma, St Louis, USA) in 0.1 M citric acid, 0.2 M, 
  NaH2PO4, pH 5.0 and 0.015% H2O2 
  as substrate; the reaction was stopped with 50 µl of 2.5 M, H2SO4. 
  Measurement of optical density (OD) at 492 nm was made in a plate reader (Merck, 
  Darmstadt, Germany).  
The cut-off value to consider a positive mouse 
  anti-core antibody response was established as twice the mean absorbance of 
  the pre-immune sera.  
To determine if human anti-HCV positive sera 
  were able to bind to Co.120 protein, a similar ELISA was carried out. Human 
  sera were individually evaluated at 1:10 dilution in PBST. Anti-human IgG-peroxidase 
  conjugate (Amersham, Little Chalfont, UK), diluted 1:3000, was employed instead 
  of anti mouse IgG-peroxidase conjugate. The other steps were performed as described 
  above. The cut-off value employed to consider a positive human anti-core antibody 
  response was established as twice the mean absorbance of the negative control 
  human sera.  
The competition of antibodies from pIDKCo plasmid-immunized 
  mice (PIM) with human anti-HCV positive sera for the binding to Co.120 protein 
  was also studied by using essentially the same ELISA. After blocking, human 
  sera at 1:10 dilution were added and incubated at 37ºC. One hour later, 
  after three washes with PBST, the plates were incubated for 1 h at 37ºC 
  with pooled sera from each group of PIM at 1:50 dilution. Anti mouse IgG-peroxidase 
  conjugate was also used at 1:3000 dilution. The inhibitory activity was expressed 
  as percentage of inhibition and determined as follows:  
Percentage of inhibition = 100 x (A492nm 
  of PIM Ab – A492nm of PIM Ab bound to Co.120 in the presence 
  of human anti-HCV positive sera)/ A492nm of PIM Ab).  
Statistical analysis - To compare differences 
  among groups, a One-way ANOVA with the Newman-Keuls post test was used. P 
  values < 0.05 were considered significant. 
 
  RESULTS 
  
Influence of additives in the anti-core antibody 
  response - BALB/c mice were immunized with the pIDKCo plasmid alone or mixed 
  with different additives. Animals were observed through 14 weeks for the development 
  of anti-HCV core Ab response. Animals vaccinated with pAEC-K6 (control plasmid) 
  did not show any reactivity against Co.120 protein when compared with their 
  own preimmune sera. In contrast, five weeks after primary immunization, anti-core 
  antibodies were detected in 80 and 100% of mice vaccinated with pIDKCo alone 
  or mixed with the additives, respectively (data not shown).  
The anti-capsid antibody response at week 5 and 
  14, determined as the OD492nm/cut-off ratio when the sera were diluted 
  1:50, is shown in Fig. 1A. The inclusion 
  of CaCl2 in the immunogen did not improve the humoral immune response. 
  On the other hand, the mixture of pIDKCo with PEG 6000, Freund's adjuvant and 
  sCT-DNA induced a statistically stronger anti-core Ab response than immunization 
  with pIDKCo alone (p < 0.01) at week 5 of the schedule. However, at week 
  14 there were not differences in the anti-core antibody response among the groups 
  of immunized mice.  
Effect of different combinations of pIDKCo 
  with Co.120 on anti-core antibody response - To investigate the effects 
  of protein-DNA combinations on the anti-core Ab response, BALB/c mice were immunized 
  with pIDKCo plasmid and Co.120 protein in different formulations. Fig. 
  1B shows the anti-core antibody response determined as OD492nm/cut-off 
  ratio when the sera were diluted 1:50.  
Five weeks after the primary immunization, all 
  groups of immunized mice had anti-core antibodies except the group vaccinated 
  with pAEC-K6. The combination of pIDKCo with Co.120 always produced a similar 
  anti-core Ab response than the protein alone, and in both groups the anti-core 
  antibody response was statistically higher than for the pIDKCo alone or the 
  prime-boost schedules (p < 0.05). There were no differences between the anti-core 
  antibody responses induced by prime/boost schedules. However, five weeks after 
  primary immunization, the priming with Co.120 and boost with pIDKCo rendered 
  statistically higher response than the group immunized with pIDKCo alone (p 
  < 0.01).  
Characterization of anti-core antibody elicited 
  by the combination of plasmid with additives or protein - Sera from mice 
  immunized with pIDKCo or Co.120 protein showed a mixed pattern of IgG2a/IgG1 
  anti-core antibodies. However, a tendency for predominance of IgG2a Abs was 
  detected in the groups immunized with prime/boost schedules or pIDKCo-PEG 6000 
  and sCT-DNA combinations (Fig. 2A-B).  

Furthermore, in order to characterize the specificity 
  of anti-core Ab response in mice, an inhibition of binding ELISA was carried 
  out (Table). Human sera were incubated 
  with Co.120 protein before incubation of mouse sera with this recombinant protein. 
  Results corresponding to sera from mice immunized with the mixture of pIDKCo 
  with CaCl2 were not analyzed because both positive and negative anti-HCV 
  human sera completely abolished the weak reactivity of these mouse sera to the 
  Co.120 protein. Positive anti-HCV human sera maximally inhibited the binding 
  of anti-core Ab induced by pIDKCo alone. Interestingly, our results showed that 
  the inclusion of additives and the combinations of plasmid with protein changed 
  the epitope specificity of anti-core antibodies. 
DISCUSSION 
  
DNA-based immunization appears promising as a 
  new way to administer vaccines. Such a vaccination strategy offers a means of 
  mimicking important characteristics of live attenuated viral vaccines like the 
  synthesis of native antigens and the induction of class I major histocompatibility 
  complex-restricted cytotoxic T lymphocyte responses. However, nucleic acid vaccines 
  do not seem to induce a response as strong as do conventional (lived attenuated) 
  vaccines and consequently different approaches have been used to modulate the 
  plasmid DNA immune responses. These efforts have been directed mainly to recruit 
  immune cells and to facilitate the entry of plasmid DNA to cells.  
Previous studies have shown that the intramuscular 
  injection of plasmid expressing the HCV core protein was capable of inducing 
  detectable core-specific antibody response, lymphoproliferative responses and 
  cytotoxic T-lymphocyte activity in mice (Major et al. 1995).  
In earlier attempts several doses of 100-200 
  µg of HCV core-encoding constructs have been generally administered in 
  mice every second week. Nevertheless, only weak humoral immune responses have 
  been induced against non-fused core variants by this way (Lagging et al. 1995, 
  Chen et al 1995, Hu et al. 1999). In contrast, we have previously demonstrated 
  that the pIDKCo plasmid induced a potent immune response following a DNA immunization 
  schedule of five doses with increasing intervals between them (Dueñas-Carrera 
  et al. 2000).  
PEG has been used to increase the efficiency 
  of yeast transfection (Beggs 1978). Besides, this molecule has been used in 
  combination with cationic lipids to increase DNA transfection in mammalian cells 
  (Harvie et al. 2000). In fact, immunization with the combination of pIDKCo and 
  PEG 6000 produced a temporary augment of the anti-core antibody response. This 
  mixture also induced a bias through IgG2a subtype, suggesting the activation 
  of cellular branch of immune response. Transfection efficiency experiments are 
  required to correlate these effects with an increased plasmid uptake in vivo. 
   
Since one of the problems of DNA immunization 
  is the degradation of plasmid by extracellular nucleases, a way to ensure cells 
  transfection is to protect the DNA from these enzymes. With this purpose we 
  employed the sonicated calf thymus DNA as a substrate competitor to diminish 
  nuclease specific degradation of the plasmid vaccine. This is the first report 
  in which sonicated calf thymus is employed with the idea of protecting plasmid 
  DNA from nucleases in vivo. Actually, we can not say that this molecule succeeded 
  in that function, but it certainly caused a transient increase in anti-core 
  antibody response when was combined with the pIDKCo plasmid. Remarkably, Weiner 
  et al. (1997) have demonstrated that calf thymus DNA, in contrast 
  to prokaryotic DNA, does not have an intrinsic adjuvant effect.  
On the other hand, calcium salts have been used 
  by Wang et al. (2000) to increase antibody titers in a formulation of a DNA 
  vaccine encoding hepatitis B surface antigen. However, mixture of CaCl2 
  with pIDKCo did not stimulate the anti-core antibody response.  

Prime-boost strategies have also been evaluated 
  to increase immune response to the HCV core antigen. Chen et al. (1995) demonstrated 
  that a second inoculation with the protein encoded by the vaccination plasmid 
  recruited lymphocyte clones primed by the first inoculation with DNA. In the 
  present work, only the prime Co.120/boost pIDKCo schedule transiently increased 
  the anti-core antibody response with respect to the generated after immunization 
  with pIDKCo alone. Moreover, the Co.120 protein alone or combined with the plasmid 
  pIDKCo induced higher anti-core antibody response than pIDKCo alone or boosted 
  with protein and vice versa. These results are consistent with the immunogenic 
  character demonstrated by Co.120 in mice and rabbits (Dueñas-Carrera 
  et al. 1999, Alvarez-Obregón et al. 2001), in spite of controversial 
  results regarding the immunogenicity of truncated nucleocapsid proteins in mice 
  (Inchauspe et al. 1997, Harase et al. 1997).  
We compared the epitope specificities of anti-core 
  antibodies in PIM with those in HCV-immune sera by an inhibitory assay. The 
  information obtained in this experiment denoted that the human HCV-immune sera 
  had antibodies that could bind to the same regions in the HCV capsid that were 
  recognized by antibodies in mice immunized with plasmid DNA. The level of inhibitory 
  activity was not related with the reactivity of anti-HCV positive human sera 
  to the capture antigen.  
Interestingly, sera from mice immunized with 
  plasmid combined with additives or Co.120 protein showed different behavior 
  in the inhibitory assay regarding those from mice immunized with pIDKCo alone. 
  Sera from mice immunized with pIDKCo evidenced the greatest inhibitory activity, 
  confirming that anti-core antibodies induced in mice by immunization with pIDKCo 
  were similar to those in human sera with respect to the epitope specificity 
  (Dueñas-Carrera et al. 2000).  
The results presented here confirmed that vaccination 
  with HCV-core derived DNA sequences could be an effective method to induce humoral 
  response to HCV. Moreover, our data indicate that kinetics, magnitude and epitope 
  specificity of the anti-HCV core humoral immune response generated following 
  DNA immunization could be modified by formulation and prime/boost strategies. 
  
ACKNOWLEDGEMENTS  
To Dr Antonieta Herrera and Dr Alejandro Martin 
  for critical and constructive reading of the manuscript.  
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  Instituto Oswaldo Cruz - Fiocruz

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