Inclusion of Vpr accessory gene in a plasmid vaccine cocktail markedly reduces Nef vaccine effectiveness in vivo resulting in CD4 cell loss and increased viral loads in rhesus macaques.

We compared the immunogenicity of plasmid vaccines containing multiple human immunodeficiency virus (HIV) antigens and found that covaccination with plasmids expressing HIV-1 14 kDa vpr gene product profoundly reduces antigen-specific CD8-mediated cytotoxic T-cell activity (CTL). Interestingly, Th1 type responses against codelivered antigens (pGag-Pol, pNef, etc.) encoded by the plasmid vaccines were suppressed. This suggested that vpr might compromise CD8 T-cell immunity in vivo during infection. A pilot primate vaccine study was designed to test the hypothesis to compare the following groups: unvaccinated controls, animals vaccinated without simean immunodeficiency virus (SIV)-Nef antigen plasmid, and animals covaccinated with the identical plasmid antigen and a plasmid construct encoding SIV Vpr/Vpx. Animals were subsequently challenged intrarectally with pathogenic SIVmac251 after the final vaccination of a multiple immunization protocol. Control animals were all infected and exhibited high viral loads and rapid CD4+ T-cell loss. In contrast, the Nef plasmid-vaccinated animals were also infected but exhibited preservation of CD4+ T-cells and a multilog reduction in viral load compared with controls. Animals covaccinated multiple times with the Nef vaccine and pVpr/Vpx plasmid suffered rapid and profound loss of CD4+ T-cells. These results have important implications for the design of multicomponent and particle vaccines for HIV-1 as well as for our understanding of HIV/SIV pathogenesis in vivo.

Issues for improving multiplasmid DNA vaccines for HIV-1.

Since the first reports of plasmid vaccines, there have been substantial changes made to the design of plasmid backbones, as well as to the antibiotic resistance markers chosen for clinical vectors compared with first generation vectors. These changes aid manufacturing, production and scale up and at the same time aid conceptual safety by limiting the ability of the vaccines to transfer useful genetic selection genes to other bacterial infectious agents. In contrast, there has been little change to the original promoters or polyadenlyation tracts in the last decade. We have learned that these first generation plasmid vaccines for HIV-1 appear very well tolerated in humans. However, while safe and immunogenic, improving the immune potency of DNA vaccines is a critical goal for this technology. The combination of antigens used should be carefully examined for possible immune interference. Such interference may only become apparent when each component of the vaccine is tested individually. This interference also suggests one mechanism of immune pathogenesis possibly by HIV-1. Optimization of the immune response can come through manipulation of the transfection efficiency, expression or through the use of various T cell and B cell plasmid adjuvants. It is likely that the combination of such advancements will significantly improve the clinical phenotype of this important vaccine modality.

Therapeutic immunization of HIV-infected chimpanzees using HIV-1 plasmid antigens and interleukin-12 expressing plasmids.

OBJECTIVE: To assess HIV-1 DNA vaccination and co-immunization with interleukin (IL)-12 and IL-10 as immunotherapy in the HIV-1 infected chimpanzee model system. METHODS: Four chimpanzees that were infected with HIV-1-IIIB for longer than 4 years and remained symptom free were immunized with HIV-1 plasmid vaccines. Two chimpanzees were immunized with DNA plasmids that encoded env/rev, gag/pol along with a plasmid that encoded both chains of human IL-12. A third animal was immunized with HIV-1 DNA vaccine constructs and co-immunized with an IL-10 expressing plasmid. Finally a control animal received the HIV-1 DNA vaccine constructs alone. RESULTS: There was no evidence of systemic toxicity associated with the administration of the DNA vaccines or the cytokine-expressing plasmids. We observed that the IL-12/HIV-1 DNA vaccinated animals had enhanced proliferative responses to multiple HIV-1 antigens at multiple time points. The animal that was co-immunized with HIV-1 and IL-10 did not have any changes in the proliferative responses. Finally, the control chimpanzee demonstrated moderate increases in the proliferative responses to HIV-1 antigens. The animal that received HIV-1 vaccines alone and the animals co-immunized with IL-12 all had declines in viral load over the course of the study, however, the decrease in viral loads were transient in all animals. CONCLUSION: Immunization of HIV-1 infected chimpanzees with DNA based vaccines containing the env, gag and pol genes can transiently boost the env specific proliferative responses. Co-administration of IL-12 expressing plasmids further leads to transient boosting of the proliferative response to the core protein, p24 as well. However, at these doses the impact on viral load is minimal.

Vaccination of seronegative volunteers with a human immunodeficiency virus type 1 env/rev DNA vaccine induces antigen-specific proliferation and lymphocyte production of beta-chemokines.

There is a pressing need to test novel vaccine concepts in an effort to develop an effective vaccine for human immunodeficiency virus (HIV) type 1. A phase I clinical study was done to test the immunogenicity of an HIV env/rev DNA vaccine, which was administered intramuscularly to HIV-1-seronegative persons. Subjects received 3 doses of vaccine at a single concentration (100 or 300 microgram) at 0, 4, 8, and 24 weeks. In at least 1 of multiple assays, the 6 subjects who received the 300-microgram dose had DNA vaccine-induced antigen-specific lymphocyte proliferative responses and antigen-specific production of both interferon-gamma and beta-chemokine. Furthermore, 4 of 5 subjects in the 300 microgram-dose group responded to both the rev and env components of the vaccine. The responses did not persist within inoculated individuals and scored in different individuals at different times in the trial. This study supports that HIV-1 DNA vaccine antigens can stimulate multiple immune responses in vaccine-naive individuals, and it warrants additional studies designed to enhance DNA vaccine immunogenicity.

HIV-1 DNA vaccines and chemokines.

DNA vaccines have a demonstrated ability to induce humoral and cellular immune responses in animal models and humans. The technology, although it dates back to the 1950's, has had an insurgence of interest within the past few years following concurrent research papers. The basic technology is being applied broadly to viral, bacterial and parasitic infections. It has also been demonstrated that genes delivered via plasmid expression vectors result in expression of functional proteins in the inoculated host. Further, injection of plasmids encoding cytokine, chemokine or co-stimulatory molecules, also referred to as immunomodulatory plasmids can lead to the further expansion of this technology to include directed immunology. We have been developing DNA technology specifically with a focus as a vaccine against HIV-1 infection. We report that such vaccines can stimulate immune responses in a variety of relevant animal systems including humoral and cellular responses as well as the production of beta-chemokines. We describe that the beta-chemokines can both modulate the immune response induced by DNA vaccines and be modulated by the DNA vaccines in the murine and chimpanzee models as well as in humans.

DNA priming-protein boosting enhances both antigen-specific antibody and Th1-type cellular immune responses in a murine herpes simplex virus-2 gD vaccine model.

It has previously been reported that herpes simplex virus (HSV)-2 gD DNA vaccine preferentially induces T-helper (Th) 1-type cellular immune responses, whereas the literature supports the view that subunit vaccines tend to induce potent antibody responses, supporting a Th2 bias. Here, using an HSV gD vaccine model, we investigated whether priming and boosting with a DNA or protein vaccine could induce both potent antibody and Th1-type cellular immune responses. When animals were primed with DNA and boosted with protein, both antibody and Th-cell proliferative responses were significantly enhanced. Furthermore, production of Th1-type cytokines (interleukin-2, interferon-gamma) was enhanced by DNA priming-protein boosting. In contrast, protein priming-DNA boosting produced antibody levels similar to those following protein-protein vaccination but failed to further enhance Th-cell proliferative responses or cytokine production. DNA priming-protein boosting resulted in an increased IgG2a isotype (a Th1 indicator) profile, similar to that induced by DNA-DNA vaccination, whereas protein priming-DNA boosting caused an increased IgG1 isotype (a Th2 indicator) profile similar to that seen after protein-protein vaccination. This result indicates that preferential induction of IgG1 or IgG2a isotype is determined by the type of priming vaccine used. Thus, this study suggests that HSV DNA priming-protein boosting could elicit both potent Th1-type cellular immune responses and antibody responses, both of which likely are important for protection against HSV infection.

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.

Engineering DNA vaccines via co-delivery of co-stimulatory molecule genes.

DNA immunization has been investigated as a potential immunization strategy against infectious diseases and cancer. To enhance a DNA vaccine's ability to induce CTL response in vivo, we co-administered CD80 and CD86 expression cassettes along with HIV-1 immunogens. This manipulation resulted in a dramatic increase in MHC class I-restricted and CD8+ T-cell-dependent CTL responses in both mice and chimpanzees. This strategy of engineering vaccine producing cells to be more efficient T-cell activators could be an important tool for optimizing antigen-specific T-cell-mediated immune responses in the pursuit of more rationally designed vaccines and immune therapies.

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.

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.

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.

Nucleic acid immunization of chimpanzees as a prophylactic/immunotherapeutic vaccination model for HIV-1: prelude to a clinical trial.

Vaccine development strategies have often utilized recombinant envelope glycoproteins which usually generate strong humoral immune responses but which do not generate strong cytotoxic T lymphocytes (CTL). A recent novel experimental vaccination approach involves the technology known as nucleic acid immunization in which DNA plasmids expressing a gene of interest is injected intramuscularly in experimental animals. These expressed proteins then are presented to the immune system with the subsequent development of strong antibody and cellular (particularly CTL) immune responses. These types of immune responses have been elicited in rodents as well as nonhuman primates including chimpanzees. Results from studies on nucleic acid immunization of HIV-1 infected chimpanzees with envelope glycoprotein expressing constructs indicated that this method was able to decrease substantially HIV-1 viral load in these chimpanzees. These data are useful for the development and implementation of human phase I clinical trials with HIV constructs expressing various genes from the HIV-1 genome.

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.

Safety and immunogenicity of intramuscular and intravaginal delivery of HIV-1 DNA constructs to infant chimpanzees.

Any global strategy for controlling the human immunodeficiency virus (HIV) epidemic is likely to rely heavily on immunization of infants and children. Given the well-documented differences in children's responses to traditional vaccines, we initiated this study to extend our findings on DNA vaccination of adult chimpanzees to immunologically immature infant chimpanzees. Our findings were consistent with our previous work in adults as we observed that the DNA vaccines used here were both well tolerated and immunogenic within weeks of the initial vaccination.