Zu Hu Huang¹, Hui Zhuang², Shan Lu³, Ren Hua Guo¹, Guo Min Xu², Jie Cai¹ and Wan Fu Zhu^2
1Department of Infectious Diseases, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
²Faculty of Microbiology, Beijing University, Beijing 100000, China
³University of Massachusetts Medical Center
Dr. Zu Hu Huang, graduated from Nanjing Medical University in 1982 and earned his Master Degree of Medicine from NMU in 1988, trained in molecular biology and virology in Australia and United States, now Professor and Director of Department of Infectious Diseases, specialized in clinical molecular biology for diagnosis and treatment of viral hepatitis, having 35 papers published.
Project supported by the grant from Science Foundation of Ministry of Health of China, No.96-1-347.
Correspondence to: Zu Hu Huang, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, Jiangsu Province, China
Telephone: 0086-25-3737024,  Fax. 0086-25-3724440
Received: 2000-08-08 Accepted: 2000-10-29

Subject headings: hepatitis B virus; DNA vaccine; hepatitis B core antigens; nucleic acid vaccine; enzyme-linked immunosorbent assay; macaca mulatta

Huang ZH, Zhuang H, Lu S, Guo RH, Xu GM, Cai J, Zhu WF. Humoral and cellular immunogenecity of DNA vaccine based on hepatitis B core gene in rhesus monkeys. World J Gastroenterol, 2001;7(1):102-106

INTRODUCTION
Hepatitis B virus (HBV) is the most common etiologic agent for infectious liver diseases. It is estimated that there are more than 250 million chronic HBV carriers in the world today and there is a significant association among persistent infection, liver cirrhosis and hepatocellular carcinoma^［1-3］. The control of HBV infections is thought to be mediated by both humoral and cellular immune responses involving neutralizing antibodies as well as class Ⅰ and class Ⅱ major histocompatibility complex (MHC) restricted T- cells^［4,5］. Among the HBV antigens, a number of studies have highlighted the importance of the human immune response against the HBcAg and HBeAg during HBV infections. During acute HBV infection, cytotoxicity T lymphocyte (CTL) specific for HBcAg and HBeAg can be detected in the circulation of the infected host. In contrast, in chronic HBV infection, HBcAg and HBeAg specific CTL and T-helper cell activity are not readily detected. The cumulated data suggest that CTL activity may play an important role in resolving HBV infection^［6-11］.
      DNA mediated immunization has been shown to be an novel method to induce both humoral and cell-mediated immune responses against many different antigens including HBV antigens^［12,13］. We have demonstrated that the DNA vaccine based on HBV core gene has strong humoral and cellular immunogenecity in different species of mice^［14,15］. In our experiments, we have further investigated the immunogenecity of this DNA vaccine in rhesus monkeys. The results show that the DNA vaccine of HBV core gene can prime obvious antigen specific antibody and cell mediated immune responses.

MATERIALS AND METHODS
Preparation of DNA vaccine of HBV core gene
The control plasmid (pJW4303) and DNA vaccine of HBV core gene (pJW4303/HBc) were propagated by a large amount of culture of the transformed E.coli strain of HB101. Plasmid DNA was purified with QIAGEN Plasmid Mega Kit (QIAGEN, Germany).

Rhesus monkeys
Four rhesus monkeys (2 male, 2 female, 3 years of age) were purchased from Special Animal Breeding and Raising Center, Xingye, Henan Province, China and maintained at the animal house in Beijing Medical University. The monkeys were divided into experimental group and control group (2 monkeys in each group).

Protocols of DNA immunization
The monkeys in the experimental group were immunized with plasmid pJW4303/HBc and that in the control group were immunized with plasmid pJW4303. The plasmids were dissolved in normal saline to a final concentration of 1g/L. Each time one monkey received 4 site intramuscular injections with a total volume of 2mL plasmid solution containing 2mg  plasmid DNA. Three boosts with same dose were given at an interval of 2 months. The monkeys’ sera before and after immunizations were collected and stored at -30℃ .

Detection of anti-HBc antibody
Anti-HBc antibodies in monkeys＇ sera were first detected by Abbott Imx System (Abbott, USA) according to the manufacturer＇s instructions and end-point titers of anti HBc antibody were then detected by an enzyme linked immunosorbent assay (ELISA). The procedures were as follows: ① The 96 well microplates were coated with recombinant HBcAg (1mg/L) and blocked with PBS containing 10% FCS. ② Three-fold dilutions of monkeys＇ sera ( 1∶50, 1∶150, 1∶450 …… 1∶984150) were added to triplicate wells. ③ HRP labeled rabbit anti-human IgG (Sino-American Biotechnology Co.) at the dilution of 1∶3000 was used as second antibody. ④ The substrate solution (TMB) was then added to each well and reaction was stopped by 2M H₂SO₄. ⑤ The absorbence value was measured at wavelength of 450nm  by an ELISA reader. Microplate washings were performed between each step with PBST solution. The end point of anti-HBc titer was defined as the highest serum dilution that resulted in an absorbence value two times that of non immune or control serum. 

Detection of IgG subclasses of anti-HBc
Subclasses of anti-HBc antibodies were detected in the sera of the monkeys positive for anti HBc. The procedures were similar to the ELISA method mentioned above for detecting anti-HBc, except that serum was diluted to 1∶30. 1∶500 diluted sheep anti human IgG1, IgG2, IgG3 and IgG4 (Nordic Immunological Laboratories, Tilburg, the Netherlands) were used as the second antibody, and 1∶5000 diluted HRP labeled rabbit anti sheep IgG (Jackson Immuno-Research Laboratories Inc, PA, USA) was used as the third antibody.

Detection of IFN-γ and IL-4 in PBMC culture supernatant
The procedures were as follows: ① PBMCs were separated from heparinized monkey blood by Ficoll gradient sedimentation method. ② PBMCs were resuspended with RPMI-1640 containing 10% FCS to a final concentration of 2×10⁶ cells/mL. ③ PBMC suspension 250μL  (5×10⁵ cells) was added to triplicate wells in a 24 well cell culture plate, and recombinant human IL-2 (500U/well) was added as well. ④ Except for control wells, PBMCs in each triplicate wells were restimulated with recombinant HBcAg at different doses of 5μg/well, 10μg/well and 12.5μg/well . ⑤ After 48h  incubation under the condition of 37℃, 5% CO₂, the supernatant was collected from each well and stored at once at -70℃. ⑥ IFN-γ and IL-4 concentrations were detected by the ELISA kits ( Jinmei Biotechnology Co., Shenzheng, China). 

PBMC proliferation assay
The procedures were similar to that for detecting IFN-γ and IL-4 in PBMC culture supernatant, except that ① PBMCs were incubated for 72h; ② 0.5μCi ³H-TdR was added to each well and followed by another 4h incubation; PBMCs were then collected onto filter membrane which were then backed 2h  at 80℃; and the radioactivity (CPM) was determined by a beta-scintillation counter (Beckman). The PBMC proliferation activity was expressed by Stimulation Index (SI), which was calculated according to the following formula: (SI=CPM of HBcAg stimulated well/CPM of non HBcAg stimulated well). SI value greater than 2 was generally considered as having antigen specific PBMC proliferation.

RESULTS
Anti-HBc IgG and its end-point titer in monkey’s sera
The results of anti-HBc  IgG and its end-point  titer in monkey’s sera are shown in Table 1.

Table 1 Anti-HBc in sera of experimental and control monkeys

^aN: negative
^b P: positive (in Abbott Imx System, the detected value less than 1.00 was considered positive for anti HBc).
^c not detected because of death.

Subclasses of anti-HBc IgG in sera of experimental group of rhesus monkeys
Subclasses of anti-HBc IgG (IgG1, IgG2, IgG3 and IgG4) were detected in the experimental monkeys (No.1 and No.2), which were found to be positive for anti-HBc in the previous tests. The results are shown in Table 2.  

Table 2 Subclasses of anti-HBc IgG and IgG1/IgG2 ratio in rhesus monkeys’sera

^a The values indicated x-±s of triplicate wells.

IFN-γ and IL-4 levels in culture supernatant of PBMCs stimulated with recombinant HBcAg
IFN-γ and IL-4 levels were detected in monkey No.2 (experimental group) and monkey No.3 (control group). Monkey No.1 and No.4 died before this test was performed. The results are shown in Table 3.

Table 3 IFN-γ and IL-4 values in culture supernatant of PBMCs

HBcAg specific PBMCs proliferation activity in experimental and control groups of rhesus monkeys
HBcAg specific PBMCs proliferation activities were measured in monkey No.2 and No.3 by the time of 12 months after first immunization. The results are listed in Table 4.

Table 4 HBcAg specific PBMC proliferation activity*

*The values in the table refer to CPM (x-±s from each triplicate well), the values in the brackets indicate stimulation index (SI).
^aP＜0.05 vs control monkey.  

DISCUSSION
DNA-mediated immunization refers to the induction of an immune response to antigen expressed in vivo subsequent to the introduction of DNA carrying the protein coding sequences and the regulatory elements needed to express them^［16,17］. An important feature of DNA-based immunization is the in situ production of the expressed protein(s), mimicking a viral infection. The endogenous synthesis should allow presentation of antigens by class Ⅰ molecules of MHC, resulting in the induction of CD8+ cytotoxic T lymphocytes (CTL)^［18］. There have been several experimented reports in which recombinant plasmid DNA was used to induce immune responses to particular pathogens, including malaria^［19］, herpes simplex virus (HSV)^［20］, influenza A^［21］, rabies virus^［22］, simian immunodeficiency virus (SIV)^［23］, human immunodeficiency virus type Ⅰ (HIV)^［24］ and hepatitis B virus (HBV)^［25-32］.
      In our earlier work, the HBV core gene fragment, which was modified to assure the high level expression of HBcAg^［33］, was successfully cloned into the plasmid pJW4303, the vector containing CMV immediate early promotor. This recombinant plasmid was named pJW4303/HBc. The DNA immunization using pJW4303/HBc among Balb/c (H₂d) and C57BL/6 (H₂b) mice showed that this recombinant could induce strong humoral (antibody) and cellular (CTL) immune responses^［34］.
      When evaluating the immunogenecity and safety of potential DNA vaccine for eventual use in humans, the nonhuman primate models should be considered. The best nonhuman primate candidate would be those closest to humans on a phylogenetic basis. However, cost and other considerations may preclude studies in hominoid species, such as chimpanzee, orang utans, gorillas, and gibbons. Based on the cost and availability, nonhominoid primate species including rhesus monkeys, represent the alternative candidate nonhuman primate species for pre-clinical immunogenecity studies^［35,36］.
      Townsend et al^［37］ observed the specific immune responses in mouse and rhesus monkeys after genetic immunization with retrovirus vectors expressing different forms of the hepatitis B virus core and e antigens. Their results showed that intramuscular injections with 10⁸ CFU of the the LHBc-Neo retrovirus vector into rhesus monkeys induced HBc/eAg specific antibody production and CD8+ CTLs. The CTL response is long lasting, and being detectable as late as 16 weeks after immunization.
     We used the plasmid as the vector to carry HBV core gene for DNA immunization in rhesus monkeys, which was different from the observation above the reason for that is that the safety of the vector for retrovirus vector was integratable to the host genome.
      In our experiments, all 4 monkeys were negative for anti-HBc before DNA immunization. After intramuscular immunization of pJW4303/HBc and pJW4303, the monkeys in the experimental group all developed anti-HBc antibody while the monkeys in the control group all negative for this antibody, indicating that this DNA vaccine could induce antigen specific humoral response in rhesus monkeys. We also found that the monkeys in the experimental group could show different antibody response profiles. Monkey No.1 became positive for anti-HBc (1∶36450) after the first immunization while monkey No.2 was not negative for anti HBc until the second immunization and the antibody titer became higher (1∶103950) as late as the total four immunizations were accomplished. This different antibody production profiles might indicate the individual difference in response to DNA immunization.
      In human and other hominoid primates, the serum IgG exhibited four subclasses, i.e. , IgG1, IgG2, IgG3 and IgG4. The relative concentrations of these IgG subclasses were 60%-70% for IgG1, 15%-20% for IgG2, 5%-10% for IgG3, and 1%-7% for IgG4. When looked into the antigen specific IgG antibodies the concentration of IgG1 and IgG2 and its ratio IgG1/IgG2 could reflect the response profiles of helper T cells (T-H1 type or T-H2 type) to some extent. Generally speaking, IgG1/IgG2＜1 or IgG1/IgG2＞1 reflected T-H1 type or T-H2 type immune responses. The previous data showed that T-H1 type response was beneficial for the clearance or eradication of chronic infected viruses while the T-H2 type- response was usually correlated to the exacerbation of immunopathogenic damage of host tissues^［38］. Feltquate et al had found that intramuscular immunization of DNA vaccines was prone to induce T-H1 type of immune response, thereby facilitating the recovery of the host from chronic viral infection^［39］. Our results also demonstrated that two monkeys intramuscularly immunized with HBV core DNA vaccine all exhibited T-H1 type of immune response based on the fact that their IgG1/IgG2 ratios were all less than 1 (0.60 and 0.58, respectively).
      The profiles of cytokine production were another indicators of helper T cell responses^［40-44］. IFN-γ, IL-2, TNF-α and GM-CSF were usually considered as T-H1 type cytokines, while IL-4, IL-5 and IL-10 were T-H2 type cytokines. IFN-γ and IL-4 were chosen in this experiment to observe helper T cell responses after DNA immunization of HBV core gene in rhesus monkeys. IFN-γ level was significantly higher in the culture supernatant of PBMC from the monkeys immunized with HBV core DNA vaccine than that from monkeys injected only with control plasmid (15.63ng/L vs ＜3.13ng/L). At the same time, IL-4 levels in both monkeys with injections of pJW4303/HBc or PJW4303 were similar (6.25ng/L vs 6.25ng/L). The results indicated IFN γ prominent cytokine profile in the monkey immunized with HBV core DNA vaccine. This result combined with the result of IgG1/IgG2 ratios mentioned above further confirmed the T-H1 type immune responses in the monkeys of the experimental group.
      Cell-mediated immune response is critical for the termination of chronic HBV infections^［45-47］. Antigen specific lymphocyte proliferation assay is an alternative for the CTL assay to evaluate the cell-mediated immune response^［48］. In this experiment, the HBcAg specific PBMC proliferation activity was seen in the monkey immunized with pJW4303/HBc but not in the monkey injected with pJW4303 (P＜0.05). After stimulation with three different doses of HBcAg, the stimulation index (SI) was all ＞2 in the experimental monkeys but all ＜2 in the control monkeys, which strongly indicated that DNA vaccine of pJW4303/HBc could induce antigen specific cell mediated immune response in rhesus monkeys.
      Sallberg et al reported that DNA immunization of HBV core gene using retrovirus as vector could markedly decrease the HBV DNA level in the sera of experimental chimpanzees, and even induce the seroconversion of HBeAg to anti-HBe^［49］. Our results showed that using plasmid as vector the DNA vaccine could also stimulate the immune responses in nonhuman primate rhesus monkeys, which was obviously helpful and beneficial for the host to inhibit and eventually eradicate chronically infected virus, including hepatitis B virus. As the designer vaccines for the 21st century, DNA vaccines demonstrated its feasibility of inducing specific cellular immunity in humans^［50］. We believed that DNA vaccine of HBV core gene may become a potential therapeutics for the treatment of chronic HBV infection in humans in the near future.

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