Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 17, Num. 2, 2000, pp. 121

Biotecnología Aplicada 2000;17:121

Immune Response against Hepatitis C Virus Core Induced
by DNA Administration Is Modulated When Additives
Are Combined with the Core Encoding Vector

Liz Álvarez-Lajonchere, Santiago Dueñas-Carrera, Lázaro J Lorenzo,
Dagmara Pichardo, Juan Morales

Centro de Ingeniería Genética y Biotecnología. PO Box 6162,
Ciudad de La Habana, Cuba.

E-mail: juan.morales@cigb.edu.cu

Code Bumber: ba00034

Introduction

DNA vaccines represent a novel means of expressing antigens in vivo for the generation of both humoral and cellular immune responses. Attempts to enhance immune responses against DNA-encoded antigens have included the combination of DNA constructs with liposomes, proteins and plasmids encoding cytokines or co-stimulatory molecules [1]. In this work, we evaluate the humoral immune response against the HCV core when different additives are combined with the DNA-encoding plasmid. We also compare protein and DNA immunization approaches in relation to the magnitude of the induced humoral immune response and the IgG2a/lgG1 antibodies ratio in the serum of immunized Balb/c mice.

Materials and Methods

Plasmids

The pAEC-K6 is an expression plasmid that contains the human cytomegalovirus immediate early promoter, the SV40 terminator, and the polyadenilation sequences and the bacterial replication origin from pUC. The pIDKCo is a pAEC-K6-derived expression vector containing the sequence encoding the first 176 aa of the HCV core protein.

Co. 120 protein

The Co. 120 is an Escherichia coli -derived protein containing the first 120 aa of HCV viral polyprotein. This HCV core protein has been estimated to be 95% pure by Coomassie staining and immunoblot analysis.

Plasmid DNA immunization

Pathogen-free Balb/c female mice of 6 to 8 weeks of age were purchased from CENPALAB (Ciudad de La Habana, Cuba) and used for all in vivo studies. Mice were injected at 0 and 3 weeks in the quadriceps muscle, with 100 mg of plasmid DNA in a 100 mL final volume of 0.9% NaCI solution. Co.120 protein was administered (10 mg per dose) in similar conditions. Serum samples were taken 5 and 14 weeks after primary immunization.

Results and Discussion

In this work, 10 groups of 5 animals were studied to analyze the effect of different additives over the anti-core specific humoral response generated after intramuscular DNA vaccination. Group 1 (control) was immunized with the pAEC-K6 plasmid. Mice were also inoculated with the pIDKCo plasmid alone (group 4) or combined with 100 mM CaCl2 (group 5), 1% PEG 6000 (group 6), Freund’s adjuvant (group 7), and 100 mg of sonicated calf thymus DNA. The truncated Co.120 protein was either evaluated alone (group 3) or in conjunction with the pIDKCo plasmid either simultaneously (group 2) or alternatively (primary immunization for group 9 and booster for group 10). Determination of anti-core specific antibodies in immunized mice was performed at 5 and 14 weeks after primary vaccination by an ELISA assay against the Co.120 protein. Although CaCI2 and PEG have been widely used to facilitate DNA incorporation in eukaryotic cells [2], it was observed a very little and transient effect on the stimulation of anti-core immune responses when combined with plasmid DNA. Something similar accounts for the combination of pIDKCo with sonicated calf thymus DNA or Freund’s adjuvant, and no statistical differences in the anti-core immune response were detected in comparison to the vector alone. However, Co.120 protein administration induced a definite stronger antibody response alone or co-injected with the pIDKCo plasmid (data not shown).

lnterestingly, the IgG2a/lgG1 ratio of anti-core antibodies in immunized mice was remarkably different. The separate immunization with plasmid DNA and protein, or their combination, rendered a mixed pattern of IgG2a/lgG1 anti-core antibodies. However, anti-core antibodies generated in all the other mice showed a clear bias towards the IgG2a subclass (Figure). In contrast, anti-core antibodies in naturally infected individuals are predominantly IgG1 [3]. Our results indicate that it is possible to manipulate the immune responses generated by DNA vaccination.

Figure. Determination of the IgG2a/lgG1 ratio in anti-core positive mouse sera. Serum samples (1:50 diluted) were pooled and evaluated for antibody subclass determination,
at 5 (open bars) and 14 (stripped bars) weeks after primary immunization, in an ELISA assay against the Co. 120 protein.

References

1. Donnelly JJ, Ulmer JB, Shiver JW, Liu MA. Annu Rev lnmunol 1997;15:617–48.

2. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning. A laboratory Manual. Second Edition. Cold Spring Harbor Laboratory, Press, New York, 1989.

3. Chen M, Sallberg M, Sonnerborg A, Weiland O, Mattsson L, Jin L, et el. Gastroenterology 1999;116(1):135–43.

Papers from Biotecnología Habana`99 Congress.
November 28-December 3, 1999.