Removing N-Terminal Sequences in Pre-S1 Domain Enhanced Antibody and B-Cell Responses by an HBV Large Surface Antigen DNA Vaccine

<div><p>Although the use of recombinant hepatitis B virus surface (HBsAg) protein vaccine has successfully reduced global hepatitis B infection, there are still a number of vaccine recipients who do not develop detectable antibody responses. Various novel vaccination approaches, including DNA vaccines, have been used to further improve the coverage of vaccine protection. Our previous studies demonstrated that HBsAg-based DNA vaccines could induce both humoral and CMI responses in experimental animal models. However, one form of the the HBsAg antigen, the large S antigen (HBs-L), expressed by DNA vaccine, was not sufficiently immunogenic in eliciting antibody responses. In the current study, we produced a modified large S antigen DNA vaccine, HBs-L(T), which has a truncated N-terminal sequence in the pre-S1 region. Compared to the original HBs-L DNA vaccine, the HBs-L(T) DNA vaccine improved secretion in cultured mammalian cells and generated significantly enhanced HBsAg-specific antibody and B cell responses. Furthermore, this improved HBsL DNA vaccine, along with other HBsAg-expressing DNA vaccines, was able to maintain predominantly Th1 type antibody responses while recombinant HBsAg protein vaccines produced in either yeast or CHO cells elicited mostly Th2 type antibody responses. Our data indicate that HBsAg DNA vaccines with improved immunogenicity offer a useful alternative choice to recombinant protein-based HBV vaccines, particularly for therapeutic purposes against chronic hepatitis infection where immune tolerance led to poor antibody responses to S antigens.</p> </div

ELISPOT analysis for IL-4 secretion in mouse splenocytes immunized with HBs-L or HBs-L(T) vaccine or empty vector.

<p>(A) Actual sample wells of IL-4 ELISPOT with mock or HBs peptide stimulation, as indicated. (B) Frequency of HBs peptide-specific IL-4 spots per million immune mouse splenocytes. The splenocytes were collected at 1 week after the last (4^th) DNA immunization. Each symbol represents an individual mouse serum sample. The geometric mean and standard deviation are shown for each group. The statistical differences between each group were determined and groups with p<0.05 are indicated.</p

Protection conferred by different H5 vaccine prime-boost regimens in ferrets.

<p>Four weeks after boost immunization, ferrets were intranasally infected with 10⁷ PFU of HK03 <i>ca</i> virus. Nasal turbinates were collected on day 3 post-infection and viral titers in tissue homogenates were determined by EID₅₀. Horizontal lines depict the mean virus titers for each group. An asterisk above a horizontal bar denotes statistical significance compared to the vector DNA group (<i>p</i><0.05, Mann-Whitney U Test).</p

HBs-specific antibody secreting cells (ASC) in splenocytes of immune mice as measured by ELISPOT.

<p>Mice were immunized with HBs-L or HBs-L(T) DNA vaccine, or with empty DNA vector as indicated. The coating antigens were anti-mouse IgG, commercial HBsAg and PBS (Mock) for detection of the total IgG, HBsAg-specific or background level ASC in mouse splenocytes. A: Actual sample wells of HBs-specific ASC spots. B: Frequency of HBs-specific ASC per 10 million splenocytes against different HBsAg coating antigens in each group. Data represent the average of spot forming cells (SFCs) per 10 million of splenocytes from each group of mice plus standard error. The splenocytes were collected 1 week after the last (4^th) DNA immunization. The statistical differences between groups were determined and difference with p<0.05 are indicated. Each symbol represents an individual mouse serum sample. The geometric mean and standard deviation are shown for each group. The statistical differences between each group were determined and groups with p<0.05 are indicated.</p

Western blot analyses of HBs-L(T), HBs-M and HBs-S proteins expressed by relevant DNA vaccines in transfected 293T cell lyate and the commercial HBs protein vaccines produced in CHO or Yeast expression system, and the empty vector transformed 293T cell lysate, as indicated.

<p>The detection antibodies were anti-S (A) or anti-Pre-S monoclonal antibodies at a concentration of 1 µg/ml.</p

ELISPOT analysis for IFN-γ secretion in mouse splenocytes immunized with HBs-L or HBs-L(T) vaccine or empty vector.

<p>(A) Actual sample wells of IFN-γ ELISPOT with mock or HBs peptide stimulation, as indicated. (B) Frequency of HBs peptide-specific IFN-γ spots per million immune mouse splenocytes. (C) The percent HBs-peptide specific CD8+ T cell responses as measured by intracellular staining. The splenocytes were collected at 1 week after the last (4^th) DNA immunization. Each symbol represents an individual mouse serum sample. The geometric mean and standard deviation are shown for each group. The statistical differences between each group were determined and groups with p<0.05 are indicated.</p

The HBsAg-specific IgG1 and IgG2a responses induced in Balb/C mice receiving DNA vaccine expressing HBs-L(T), HBs-M and HBs-S proteins, the commercial HBs protein vaccines produced in CHO or Yeast expression system, as indicated.

<p>The data represent the geometric mean of IgG2a/IgG1 ratios with standard deviations of each group of 5 mice.</p

Schematic design of HBs-L DNA vaccines.

<p>A. These HBs-L DNA vaccines express the full length large protein of the HBsAg (HBs-L) or the truncated large protein with removal of the first 18 amino acids at the N-terminus, respectively. The amino acid positions for each HBs-L immunogen are as indicated. B and C: The ELISA analyses of the HBs-L and HBs-L(T) protein expressions by the relevant DNA and the empty vector as negative control, using commercially available anti-S (B) or anti-Pre-S mAb (C). “S” and “L” represent the supernatant and lysate of transiently transfected 293T cells.</p

HBs-specific antibody responses in Balb/C mice receiving DNA vaccine expressing HBs-L or HBs-L(T), or the empty vector as negative control.

<p>A: Temporal HBs-specific antibody responses in immune mouse sera (1∶500 dilution) detected against commercial HBsAg antigen. The arrows show the time points of DNA immunization. The OD values are expressed as the average of group. B and C: HBs-specific IgG titers (reciprocal serum dilution) induced by HBs-L or HBs-L(T) DNA vaccine or empty vector were detected against commercial HBsAg antigen (B) by ELISA or by a commercial HBs-specific antibody detection kit. Antibody titers were measured using sera collected at 2 weeks after the 3^rd DNA immunization. Each symbol represents an individual mouse serum sample. The geometric mean and standard deviation are shown for each group. The statistical differences between each group were determined and groups with p<0.05 are indicated.</p

H5 HA DNA vaccine construct.

<p>(A) Schematic diagram of H5-VN.tPA HA gene. The H5-VN.tPA DNA encodes the full length HA antigens with a human tissue plasminogen activator (tPA) leader substituting for the natural HA leader sequence. The cleavage site between HA1 and HA2 subunits are indicated. The numbers above the HA inserts denote the relevant amino acid positions in natural HA proteins. (B) Western-blot analysis of the expression of HA antigen by H5-VN.tPA DNA vaccine in transiently transfected 293T cell supernatant (S) and cell lysate (L).</p