Margaret A. Liu

Generation of MHC class I‐restricted cytotoxic T lymphocytes by expression of a viral protein in muscle cells: antigen presentation by non‐muscle cells

Expression of reporter genes in muscle cells has been achieved by intramuscular (i.m.) injection of plasmid DNA expression vectors. We previously demonstrated that this technique is an effective means of immunization to elicit both antibodies capable of conferring homologous protection and cell‐mediated immunity leading to cross‐strain protection against influenza virus challenge in mice. These results suggested that expression of viral proteins by muscle cells can result in the generation of cellular immune responses, including cytotoxic T lymphocytes (CTL). However, because DNA has the potential to be internalized and expressed by other cell types, we sought to determine whether or not induction of CTL required synthesis of antigen in non‐muscle cells and, if not, whether transfer of antigen to antigen‐presenting cells from muscle cells may be involved. In the present study, we demonstrate that transplantation of nucleoprotein (NP)‐transfected myoblasts into syngeneic mice led to the generation of NP‐specific antibodies and CTL, and cross‐strain protective immunity against a lethal challenge with influenza virus. Furthermore, transplantation of NP‐expressing myoblasts (H‐2k) intraperitoneally into F1 hybrid mice (H‐2du2003×u2003H‐2k) elicited NP CTL restricted by the MHC haplotype of both parental strains. These results indicate that NP expression by muscle cells after transplantation was sufficient to generate protective cell‐mediated immunity, and that induction of the CTL response was mediated, at least in part, by transfer of antigen from the transplanted muscle cells to a host cell.

Protective immunity by intramuscular injection of low doses of influenza virus DNA vaccines

Dose-response relationships were investigated between dose of influenza virus haemagglutinin (HA) or nucleoprotein (NP) DNA vaccines, and immunogenicity and protective efficacy based on humoral and cellular immunity. In mice, intramuscular (i.m.) injection of HA or NP DNA, at doses of 100 ng to 1 microgram, was found to generate haemagglutination inhibiting (HI) antibodies and cytotoxic T-lymphocytes, respectively, and provide protection in influenza virus challenge models. A direct correlation between the amount of DNA injected and the level of HI antibody was observed. In non-human primates, high-titre neutralizing antibodies were induced in animals vaccinated with as little as 10 micrograms of HA DNA. These results indicate that low doses of DNA administered by i.m. injection provide protective efficacy against influenza.

Characterization of humoral immune responses induced by an influenza hemagglutinin DNA vaccine

We have examined in detail the characteristics of the humoral immune response and protective efficacy induced by an influenza hemagglutinin (HA) DNA vaccine. In mice injected intramuscularly with HA DNA, the magnitude of the immune responses generated, as measured by ELISA and hemagglutination inhibiting (HI) antibodies, was directly related to the amount of DNA injected and the number of doses administered. The level of anti-HA antibodies in DNA-vaccinated mice was higher than that in convalescent immune mice and was maintained for at least 1.5 years. The immunoglobulin isotype profile of the antibodies was predominantly IgG2a, similar to that induced by live virus infection but in contrast to the relative abundance of IgG1 antibodies observed after inoculation with formalin-inactivated whole virus. The presence of pre-challenge HI antibodies was found to be a good correlate of protection, in that every animal with a detectable HI titer was protected from a lethal challenge. Complete protection from a lethal dose of influenza virus (A/PR/34), as judged by 100% survival and no weight loss, was conferred by as little as 1 microgram of DNA (given twice). Furthermore, mice injected with 10 to 100 micrograms doses, when subsequently challenged with virus, showed no increase in HI titer and no production of antibodies directed against the challenge virus, suggesting a substantial inhibition of virus replication after challenge.

Further protection against antigenic drift of influenza virus in a ferret model by DNA vaccination

Previously we showed that immunization of ferrets with DNA encoding the hemagglutinin (HA), nucleoprotein (NP), and matrix protein (M1) of influenza virus induced protective immune responses. A DNA vaccine encoding HA (from a 1991 strain), NP and M1 (from a 1989 strain) protected ferrets better against challenge with the antigenic drift variant A/Georgia/03/93 than did the inactivated vaccine from the 1992-93 influenza season. Here we report that the same DNA vaccine protected ferrets against a second, further divergent, drift variant (A/Johannesburg/33/94). Furthermore, the extent of protection provided by the DNA vaccine was equivalent to the homologous protection provided by an inactivated vaccine that exactly matched the challenge strain.

Immunization of non-human primates with DNA vaccines

The pre-clinical efficacy of DNA vaccines has been demonstrated in a number of animal models, but more limited data exist regarding their immunogenicity in non-human primates. The studies described below demonstrate that DNA vaccines in reasonable dosages encoding a variety of viral proteins could result in the generation of antibodies, neutralizing antibodies, or cytotoxic T lymphocytes in primates. Furthermore, these responses could be boosted by repeat administration of the DNA vaccine. In an effort to assess the safety of such vaccines sera from primates was shown to lack anti-DNA antibodies.

Induction of MHC class I-restricted CTL response by DNA immunization with ubiquitin-influenza virus nucleoprotein fusion antigens

DNA vaccines have been shown to be an effective means of inducing cytotoxic T-lymphocyte (CTL) responses in both young and aged mice. Better understanding of the pathways by which antigens encoded by DNA vaccines are processed and presented to CTL may allow for improvements in CTL responses in older animals. Since CTL recognize short peptides presented by MHC class I molecules, and since ubiquitin-dependent proteolysis is widely believed to be responsible for degradation of endogenously synthesized antigens and generation of these peptide ligands, we sought to use ubiquitin (Ub) conjugation to target influenza virus nucleoprotein (NP) antigen into the Ub-proteasome degradation pathway for MHC class I-restricted antigen processing and presentation. However, the addition of the Ub moiety did not affect the half-life of Ub-NP protein in transiently transfected human rhabdomyosarcoma (RD) cells. Moreover, the modifications of NP DNA vaccine with Ub conjugation did not affect their ability to induce a CTL response specific for the H-2Kd-restricted NP147-155 epitope, as assessed by both percent cytolysis in bulk CTL culture and by CTL precursor (CTLp) frequency in limiting dilution analysis (LDA). In contrast, the anti-NP antibody (Ab) responses were dramatically reduced in mice immunized with low doses (1 microgram) of Ub-NP constructs, compared with mice immunized with wild-type NP DNA. These results demonstrate that Ub conjugation alone does not guarantee targeting of endogenously synthesized antigens for rapid degradation by proteasomes. Furthermore, the ability of ubiquintination to reduce Ab responses to NP without affecting CTL responses suggests that the Ub modifications result in a lower availability of full-length NP from transfected cells in vivo. The implications of these data on antigen presentation and cross-priming are discussed.

Expression and immunogenicity of Mycobacterium tuberculosis antigen 85 by DNA vaccination

Plasmid DNA expression vectors encoding Mycobacterium tuberculosis antigen 85 (Ag85) were tested as vaccines in preclinical animal models. Expression of secreted and nonsecreted forms of Ag85 was observed after transient transfection of cells in vitro. In mice, both types of Ag85 DNA constructs induced strong humoral and cell-mediated immune responses, as measured by ELISA of sera and recall responses of spleen cells restimulated in vitro, respectively, Therefore, DNA vaccination is an effective means of expressing mycobacterial proteins in eukaryotic cells leading to the induction of potent immune responses.

Cytotoxic T Lymphocyte and Helper T Cell Responses following HIV Polynucleotide Vaccination

Expression vectors encoding either HIV-1 gp160/rev, gp120, or rev alone were used for direct vaccination of mice and nonhuman primates. Each vaccine elicited long-lived (> 7 months) helper T cell responses in mice and monkeys as measured by in vitro proliferation of splenocytes following recombinant antigen treatment. Cytokine assays of the cell supernatants showed that approximately 100-fold more gamma-interferon than IL-4 was secreted during culture indicating that these vaccines elicited TH1-like responses. CD8+ CTL activities were also observed both in mice and rhesus. The gp120 and gp160/rev vaccines elicited antigen-specific antibodies, although these responses were more variable and lower magnitude for gp160/rev, and gp120 DNA-vaccinated African green monkeys had moderate levels of neutralizing antibodies. No antibodies were found against rev (an intracellular protein) with either rev vaccine. Similar antibody titers were obtained for gp120 by either intramuscular or intradermal injection although T cell responses were generally lower by intradermal route. These results indicate that DNA vaccines may provide a powerful means to elicit cellular and humoral immune responses against HIV.

Anti-HIV env immunities elicited by nucleic acid vaccines

Plasmid DNA vaccines encoding HIV-1 env were used to immunize mice and nonhuman primates. Plasmids were prepared that produced either secreted gp120 or full-length gp160. Mice immunized with gp120 DNA developed strong antigen-specific antibody responses, CD8+ cytotoxic T lymphocytes (CTL) (following in vitro restimulation with gp120-derived peptide), and showed in vitro proliferation and Th1-like cytokine secretion [gamma-interferon, interleukin (IL)-2 with little or no IL-4] by lymphocytes obtained from all lymphatic compartments tested (spleen, blood, and inguinal, iliac, and mesenteric lymph nodes). This indicated that systemic anti-gp120 cell-mediated immunity was induced by this DNA vaccine. Although similar antibody responses were observed in mice immunized by either intramuscular or intradermal routes, T cell responses were significantly stronger in mice injected intramuscularly. Rhesus monkeys immunized with both gp120 and gp160 DNAs exhibited significant CD8+ CTL responses, following in vitro restimulation of peripheral blood lymphocytes with antigen. These experiments demonstrate that DNA immunization elicits potent immune responses against HIV env in both a rodent and a nonhuman primate species.

Expression of a viral protein by muscle cells in vivo induces protective cell-mediated immunity

Intramuscular injection of plasmid DNA expression vectors results in transfection of myocytes in situ. To determine whether expression of antigen by myocytes is sufficient to induce protective cell-mediated immunity, stably transfected myoblasts expressing influenza nucleoprotein (NP) were transplanted into mice. These animals produced high-titer anti-NP antibodies and MHC class I-restricted cytotoxic T lymphocytes, and were protected from a cross-strain lethal challenge with influenza A virus. Therefore, antigen expression by muscle cells in vivo is sufficient to confer protective cell-mediated immunity.