Anti-inflammatory cytokines, pro-fibrogenic chemokines and persistence of acute HCV infection.

Chemokines and cytokines play a vital role in directing and regulating immune responses to viral infections. Persistent hepatitis C virus (HCV) infection is characterized by the loss of anti-HCV cellular immune responses, while control of HCV infection is associated with maintenance of anti-HCV cellular immune responses. To determine whether plasma concentrations of 19 chemokines and cytokines controlling T-cell trafficking and function differed based on infection outcome, we compared them in at-risk subjects followed prospectively for HCV infection. Levels were compared over time in subjects who controlled HCV infection (Clearance) and subjects who developed persistent HCV infection (Persistence) at two time points during acute infection: (i) first viraemic sample (initial viraemia) and (ii) last viraemic sample in Clearance subjects and time-matched samples in Persistence subjects. At initial viraemia, increased pro-inflammatory tumour necrosis factor α (TNFα) plasma concentrations were observed in the Clearance group, while the plasma levels of anti-inflammatory interleukin (IL)-2, IL-10 and IL-13 were higher in the Persistence group. IL-13 was positively correlated with IL-2 and IL-10 at initial viraemia in the Persistence group. At the time of last viraemia, plasma levels of eotaxin, macrophage chemoattractant protein-4 (MCP-4), IL-5 and IL-10 were higher in the Persistence group and IL-10 and IL-5 levels were positively correlated. Collectively, these results suggest that the development of persistent infection is associated with an anti-inflammatory and pro-fibrogenic chemokine and cytokine profile that is evident at the onset of infection and maintained throughout acute infection.

Plasmids encoding the mucosal chemokines CCL27 and CCL28 are effective adjuvants in eliciting antigen-specific immunity in vivo.

A hurdle facing DNA vaccine development is the ability to generate strong immune responses systemically and at local immune sites. We report a novel systemically administered DNA vaccination strategy using intramuscular codelivery of CCL27 or CCL28, which elicited elevated peripheral IFN-gamma and antigen-specific IgG while driving antigen-specific T-cell secretion of cytokine and antibody production in the gut-associated lymphoid tissue and lung. This strategy resulted in induction of long-lived antibody responses that neutralized influenza A/PR8/34 and protected mice from morbidity and mortality associated with a lethal intranasal viral challenge. This is the first example of the use of CCL27 and CCL28 chemokines as adjuvants to influence a DNA vaccine strategy, suggesting further examination of this approach for manipulation of vaccine-induced immunity impacting both quality and phenotype of responses.

Targeted antigen delivery to antigen-presenting cells including dendritic cells by engineered Fas-mediated apoptosis.

Immunity to tumors as well as to viral and bacterial pathogens is often mediated by cytotoxic T lymphocytes (CTLs). Thus, the ability to induce a strong cell-mediated immune response is an important requirement of novel immunotherapies. Antigen-presenting cells (APCs), including dendritic cells (DCs), are specialized in initiating T-cell immunity. Harnessing this innate ability of these cells to acquire and present antigens, we sought to improve antigen presentation by targeting antigens directly to DCs in vivo through apoptosis. We engineered Fas-mediated apoptotic death of antigen-bearing cells in vivo by co-expressing the immunogen and Fas in the same cell. We then observed that the death of antigen-bearing cells results in increased antigen acquisition by APCs including DCs. This in vivo strategy led to enhanced antigen-specific CTLs, and the elaboration of T helper-1 (Th1) type cytokines and chemokines. This adjuvant approach has important implications for viral and nonviral delivery strategies for vaccines or gene therapies.

Engineering of DNA vaccines using molecular adjuvant plasmids.

These studies support the view that additional goals of enhancing DNA vaccine technology will probably be at several levels. The ability to deliver antigens more efficiently to professional APCs is likely to have important implications for our studies of basic principles of immunology. Furthermore, there are simple practical approaches to vaccine enhancement that can be tested with the present group of DNA vaccines. These studies should include the use of cytokine molecular adjuvants as well as possible co-stimulatory molecules. It is expected that the delivery of these "adjuvanted" DNA vaccines will require additional safety evaluation; however, it is clear that studies can be easily designed to address the important safety issues associated with these novel vaccine adjuvants. Overall, the results indicate that further more precise quantitative studies and combination studies examining these additional promising adjuvant candidates are warranted.

Systemic and mucosal immunity is elicited after both intramuscular and intravaginal delivery of human immunodeficiency virus type 1 DNA plasmid vaccines to pregnant chimpanzees.

DNA vaccines encoding human immunodeficiency virus type 1 (HIV-1) env/rev and gag/pol were delivered intravaginally (IVAG) and intramuscularly (IM) to 2 pregnant chimpanzees. Vaccination was well tolerated and each chimpanzee developed antibodies (up to 1 year later) to both vaccines. Placental transfer of anti-Env and anti-Gag IgG was demonstrated in both maternal/infant pairs. Specific IgG was also demonstrated in saliva, vaginal, and rectal washes after IVAG immunization. Predominantly anti-HIV-1 IgA was detected in the milk of both mothers after both IM and IVAG immunization. Cellular responses included Gag-specific proliferation of lymphocytes and cytotoxic T lymphocytes against both antigens. These data suggest a strategy for induction of mucosal and systemic responses after both IM and IVAG delivery of DNA vaccines in a primate model and could ultimately be useful in lowering maternal-to-fetal transmission of HIV-1, perinatally and through breastfeeding.

Enhancement of cellular immune response in HIV-1 seropositive individuals: A DNA-based trial.

A DNA-based vaccine containing HIV-1 Env and Rev genes was tested for safety and host immune response in 15 HIV-infected asymptomatic patients with CD4-positive lymphocyte counts >/=500/microl of blood and receiving no antiviral therapy. Successive groups of patients received three doses of vaccine at 30, 100, or 300 microg at 10-week intervals in a dose-escalation trial. Some changes were noted in cytotoxic T-lymphocyte activity against gp160-bearing targets. Importantly, enhanced specific lymphocyte proliferative activity against HIV-1 envelope was observed in multiple patients. Three of three patients in the 300-microg dose group also developed increased MIP-1alpha levels which were detectable in their serum. Interestingly patients in the lowest dose group showed no overall changes in the immune parameters measured. The majority of patients who exhibited increases in any immune parameters were contained within the 300 microg, which was the highest dose group. These studies support further investigation of this technology for the production of antigen-specific immune responses in humans.

HIV-1 DNA based vaccine induces a CD8 mediated cross-clade CTL response.

Novel approaches for the generation of more effective vaccines for HIV-1 are of significant importance. In this report we analyse the immunogenicity and efficacy of a DNA vaccination approach in a chimpanzee model system. Three chimpanzees were vaccinated with DNA constructs which express the env, rev, gag and pol proteins. These animals developed specific cellular responses to these proteins, although the nature of the responses varied among the animals. We demonstrated that DNA vaccination led to a CD8 mediated killing of targets expressing the homologous clade B envelope as well as targets expressing heterologous clade E envelope. In addition seronegative individuals have been inoculated with a DNA construct which expresses the env, rev proteins. These studies serve as an important benchmark for the use of DNA vaccine technology for the production of protective immune responses.

DNA vaccination with HIV-1 expressing constructs elicits immune responses in humans.

Humoral and cellular immune responses have been produced by intramuscular vaccination with DNA plasmids expressing HIV-1 genes, suggesting possible immunotherapeutic and prophylactic value for these constructs. Vaccination with these constructs has decreased HIV-1 viral load in HIV-1-infected chimpanzees. In addition, naive (i.e. non-HIV-1-infected) chimpanzees were protected against a heterologous challenge with HIV-1. Ongoing phase I clinical trials show that therapeutic vaccinations indeed boost anti-HIV-1 immune responses in humans. A therapeutic phase I trial on humans with these constructs induced a good safety profile and also demonstrated an immunological potentiation. These findings indicate that further studies with these constructs in humans are warranted.

Safety and immunogenicity of HIV-1 DNA constructs in chimpanzees.

A global effort to control the HIV epidemic is likely to rely heavily on immunization strategies. As our closest genetic relative, the chimpanzee provides the most important model for preclinical safety and immunogenicity studies. We have immunized adult, pregnant and infant chimpanzees with our plasmid vaccines. We have found these vaccines to be safe and well tolerated in all of these groups. The same vaccines have induced both humoral and cellular immunity in each instance.

First human trial of a DNA-based vaccine for treatment of human immunodeficiency virus type 1 infection: safety and host response.

A DNA-based vaccine containing human immunodeficiency virus type 1 (HIV-1) env and rev genes was tested for safety and host immune response in 15 asymptomatic HIV-infected patients who were not using antiviral drugs and who had CD4+ lymphocyte counts of > or = 500 per microliter of blood. Successive groups received three doses of vaccine (30, 100, or 300 microg) at 10-week intervals in a dose-escalation trial. Vaccine administration induced no local or systemic reactions, and no laboratory abnormalities were detected. Specifically, no patient developed anti-DNA antibody or muscle enzyme elevations. No consistent change occurred in CD4 or CD8 lymphocyte counts or in plasma HIV concentration. Antibody against gp120 increased in individual patients in the 100- and 300-/microg groups. Some increases were noted in cytotoxic T lymphocyte activity against gp160-bearing targets and in lymphocyte proliferative activity. The safety and potential immunogenicity of an HIV-directed DNA-based vaccine was demonstrated, a finding that should encourage further studies.

Specific immune induction following DNA-based immunization through in vivo transfection and activation of macrophages/antigen-presenting cells.

The initiation of an adaptive immune response requires Ag presentation in combination with the appropriate activation signals. Classically, Ag presentation and immune activation occur in the lymph node and spleen, where a favorable organ architecture and rich cellular help can enhance the process. Recently, several investigators have reported the use of DNA expression cassettes to elicit cellular and humoral immunity against diverse pathogens. Although the immune mechanisms involved are still poorly understood, plasmid inoculation represents a model system for studying immune function in response to invading pathogens. In this report, we demonstrate the presence of activated macrophages or dendritic cells in the blood lymphocyte pool and peripheral tissues of animals inoculated with DNA expression cassettes. These cells are directly transfected in vivo, present Ag, and display the surface proteins CD80 and CD86. Our studies indicate that these cells function as APC and can activate naive T lymphocytes. They may represent an important first step APC in genetic immunization and natural infection.

Induction of a TH1 type cellular immune response to the human immunodeficiency type 1 virus by in vivo DNA inoculation.

DNA inoculation is capable of producing antigens intracellularly for ultimate presentation to the cellular and humoral components of the immune system and has potential for vaccine strategies against a number of infectious pathogens including HIV-1. It is well documented that the antigenic diversity of HIV-1 and its high level of nucleotide mutations during reverse transcription can lead to escape from immune surveillance. However, data suggest that a CD8-mediated cytotoxic T lymphocyte response may be less susceptible to escape mutants. We have shown previously that in vivo inoculation of rodents and non-human primates with plasmid expression vectors encoding HIV-1 gene products leads to production of HIV-1 antigens and results in the production of both cellular and humoral immune responses. In addition we have also demonstrated previously that these responses lead to protection in several in vivo models. We further demonstrate here that the cellular response induced is a type TH1 response and specific lysis of HIV-infected targets is CD8-mediated.

Monoclonal antibodies define a cellular antigen involved in HTLV-I infection.

The exact mechanism by which the human T cell leukemia viruses (HTLV) infects target cells remains unclear; although some molecules have been identified to be important in viral infection and entry. To investigate these phenomena, we generated a panel of monoclonal antibodies (MAb) against a B cell line (BJAB-WH) which is highly permissive for infection with HTLV. These MAb have been used to further characterize the membrane molecules important for HTLV infection. Three of these MAb designated 4.2.3, 3.3.10, and 11.2.3 were capable of inhibiting syncytium formation induced in human B and T cell lines (i.e., BJAB-WH and SupT-1, respectively) by co-culture with HTLV-I infected MT-2 cells. All of these MAbs immunoprecipitated a 80-85 kDa antigen from the lysates of metabolically labeled BJAB-WH but not from BJAB-CC/84, a noninfectible target cell. The binding of these MAb with different HTLV target cells was analyzed and compared with binding of polyclonal monospecific antisera to the same cell lines. A 80-85 kDa membrane glycoprotein was isolated with an immunoaffinity chromatographic column constructed with MAbs 4.2.3 and 3.3.10. This cellular antigen was capable of inhibiting HTLV I/MT-2 induced fusion. This is the first direct demonstration that a 80-85 kDa cellular glycoprotein is directly involved in HTLV I/II infection and syncytium formation.

Modulation of amplitude and direction of in vivo immune responses by co-administration of cytokine gene expression cassettes with DNA immunogens.

Immunization with nucleic acids has been shown to induce both antigen-specific cellular and humoral immune responses in vivo. We hypothesize that immunization with DNA could be enhanced by directing specific immune responses induced by the vaccine based on the differential correlates of protection known for a particular pathogen. Recently we and others reported that specific immune responses generated by DNA vaccine could be modulated by co-delivery of gene expression cassettes encoding for IL-12, granulocyte-macrophage colony-stimulating factor and the co-stimulatory molecule CD86. To further engineer the immune response in vivo, we investigated the induction and regulation of immune responses following the co-delivery of pro-inflammatory cytokine (IL-1 alpha, TNF-alpha, and TNF-beta), Th1 cytokine (IL-2, IL-12, IL-15, and IL-18), and Th2 cytokine (IL-4, IL-5 and IL-10) genes. We observed enhancement of antigen-specific humoral response with the co-delivery of Th2 cytokine genes IL-4, IL-5, and IL-10 as well as those of IL-2 and IL-18. A dramatic increase in antigen-specific T helper cell proliferation was seen with IL-2 and TNF-alpha gene co-injections. In addition, we observed a significant enhancement of the cytotoxic response with the co-administration of TNF-alpha and IL-15 genes with HIV-1 DNA immunogens. These increases in CTL response were both MHC class I restricted and CD8+ T cell dependent. Together with earlier reports on the utility of co-immunizing using immunologically important molecules together with DNA immunogens, we demonstrate the potential of this strategy as an important tool for the development of more rationally designed vaccines.

Molecular and immunological analysis of genetic prostate specific antigen (PSA) vaccine.

Nucleic acid immunization has been investigated as immunotherapy for infectious diseases as well as for treating specific types of cancers. In this approach, nucleic acid expression cassettes are directly inoculated into the host, whose transfected cells become the production source of novel and possibly immunologically foreign protein. We have developed a DNA vaccine construct which encodes for PSA by cloning a cDNA for PSA into a mammalian expression vector under control of a CMV promoter. We investigated and characterized the immunogenicity of PSA DNA expression cassettes in mice. PSA-specific immune responses induced in vivo by immunization were characterized by enzyme-linked immunosorbent assay (ELISA), T helper proliferation cytotoxic T lymphocyte (CTL), and flow cytometry assays. We observed a strong and persistent antibody response against PSA for at least 180 days following immunization. In addition, a significant T helper cell proliferation was observed against PSA protein. Using synthetic peptides spanning the PSA open frame, we identified four dominant T helper epitopes of PSA. Furthermore, immunization with PSA plasmid induced MHC Class I CD8+ T cell-restricted cytotoxic T lymphocyte response against tumor cell targets expressing PSA. The prostate represents a very specific functional organ critical for reproduction but not for the health and survival of the individual. Understanding the immunogenicity of PSA DNA immunization cassettes offers insight into the possible use of this tumor-associated antigen as a target for immunotherapy. These results demonstrate the ability of the genetic PSA to serve as a specific immune target capable of generating both humoral and cellular immune responses in vivo.

DNA vaccination as anti-human immunodeficiency virus immunotherapy in infected chimpanzees.

The role of the immune response in controlling human immunodeficiency virus type 1 (HIV-1) replication is controversial. Immunotherapeutic strategies that have the ability to broaden immune responses might play a role in slowing disease progression. DNA immunization was studied as immunotherapy in infected chimpanzees. Two HIV-1-infected chimpanzees were vaccinated with DNA plasmid vaccines, one with plasmid pCMN160, which expresses the envelope glycoprotein of HIV-1MN and rev, and the other with a control plasmid. The chimpanzee immunized with pCMN160 demonstrated enhanced humoral responses. Virus load was monitored. Virus load in the chimpanzee receiving pCMN160 decreased at week 20 and has remained at background levels. The control chimpanzee was subsequently vaccinated with pCMN160. After immunization, the antibody responses increased and, as in the first animal, the virus load decreased. These results indicate the potential of the immune response to have a direct impact on HIV-1 replication in chimpanzees.

Genetic immunization: a new era in vaccines and immune therapeutics.

DNA inoculation represents a novel approach to vaccine and immune therapeutic development. The direct injection of gene expression cassettes into a living host transforms a number of cells into factories for production of the introduced gene products. Expression of these delivered genes has important immunological consequences and may result in the specific immune activation of the host against the novel expressed antigens. The recent demonstration by laboratories that these immune responses are protective in some infectious disease experimental models as well as cancers is viewed with cautious optimism. Further, the relatively short development times, ease of large-scale production, low development, manufacturing, and distribution costs all combine with immunological effectiveness to suggest that this technology will dramatically influence the production of a new generation of experimental vaccines and immune therapies. It is hoped that DNA inoculation will ultimately lead to new vaccines that are immunologically effective and economically accessible to all nations.

Engineering of in vivo immune responses to DNA immunization via codelivery of costimulatory molecule genes.

Nucleic acid immunization is a novel vaccination technique to induce antigen-specific immune responses. We have developed expression cassettes for cell surface markers CD80 and CD86, two functionally related costimulatory molecules that play an important role in the induction of T cell-mediated immune responses. Coimmunization of these expression plasmids, along with plasmid DNA encoding for HIV-1 antigens, did not result in any significant change in the humoral response; however, we observed a dramatic increase in cytotoxic T-lymphocyte (CTL) induction as well as T-helper cell proliferation after the coadministration of CD86 genes. In contrast, coimmunization with a CD80 expression cassette resulted in a minor, but positive increase in T-helper cell or CTL responses. This strategy may be of value for the generation of rationally designed vaccines and immune therapeutics.

Development of a multicomponent candidate vaccine for HIV-1.

Nucleic acid or DNA immunization represents a novel approach to both vaccine and immune therapeutic development. DNA vaccination induces antigen-specific cellular and humoral immune responses through the delivery of non-replicating transcription units which drive the synthesis of specific foreign proteins within the inoculated host. We have previously reported on the potential use of DNA immunization as a novel vaccine strategy for HIV-1. We found that both antigen-specific cellular and humoral immune responses could be induced in vivo with various DNA vaccine constructs against different antigenic targets within HIV-1. In order to enhance the DNA vaccine's ability to elicit cell-mediated immune responses, we co-delivered plasmids encoding costimulatory molecule B7 and interleukin-12 genes with DNA vaccine for HIV-1. We observed a dramatic increase in both antigen-specific T helper cell proliferation and CTL response. Eventual development of successful vaccines for HIV-1 would likely involve targeting multiple antigenic components of the virus to direct and empower the immune system to protect the host from viral infection. We present here the utility of multicomponent DNA immunization to elicit specific humoral and cell-mediated immune responses against different antigenic targets of HIV-1 as well as the ability of this immunization strategy to achieve significant enhancements of antigen-specific cellular immune responses.

Protection of chimpanzees from high-dose heterologous HIV-1 challenge by DNA vaccination.

Novel approaches for the generation of more effective vaccines for HIV-1 are of significant importance. In this report we analyze the immunogenicity and efficacy of an HIV-1 DNA vaccine encoding env, rev and gag/pol in a chimpanzee model system. The immunized animals developed specific cellular and humoral immune responses. Animals were challenged with a heterologous chimpanzee titered stock of HIV-1 SF2 virus and followed for 48 weeks after challenge. Polymerase chain reaction coupled with reverse transcription (RT-PCR) results indicated infection in the control animal, whereas those animals vaccinated with the DNA constructs were protected from the establishment of infection. These studies serve as an important benchmark for the use of DNA vaccine technology for the production of protective immune responses.