Fred C. Jensen

Characterization of highly purified, inactivated HIV-1 particles isolated by anion exchange chromatography

This report characterizes inactivated, gp120 depleted, HIV-1 particles purified by an anion exchange chromatography production process. This antigen formulated with incomplete Freunds adjuvant constitutes Remune, which is being evaluated in a phase III clinical endpoint trial to determine the effect of this immune-based therapy on clinical progression of HIV-1 seropositive patients. Multiple production lots of the inactivated HIV-1 antigen strain HZ321, isolated by anion exchange chromatography, exhibit purity of > 95% by gel filtration. These findings are corroborated by thin section electron microscopy showing a homogenous field of intact particles. Analyses of the purified virus particles for protein, lipid, carbohydrate and RNA show structural retention of the envelope proteins, lipid bilayer and core components after large scale processing. The qualitative identification of at least 85% of total HIV-1 protein is determined by ELISA, Western blot, HPLC and amino acid sequencing analyses. Quantitative values are assigned to 50% of these proteins. The data confirm the presence of virally encoded proteins p6, p7, pI15, p17, p24, p32, pI39Gag, gp41, pp55Gag, p66/51, Vpr, Vif and Nef. Excellent consistency between production lots and equivalency to HIV-1 preparations purified by sucrose density gradient sedimentation has been established for protein and lipid composition, and overall purity. These findings further establish that non-viral encoded proteins and lipids are integral structural components of the intact virion and are not contaminants unique to a particular isolation method. The data confirm the presence of multicomponent antigens in the viral particles for stimulating a broad HIV-1 specific immune response. Finally, the work demonstrates that the two inactivation procedures (beta-propiolactone and gamma irradiation), which achieve efficient viral inactivation meeting US FDA guidelines, do not damage the protein antigens of the viral particles.

In vitro immune function after vaccination with an inactivated, gp120-depleted HIV-1 antigen with immunostimulatory oligodeoxynucleotides

We examined the effect of a synthetic oligodeoxynucleotide containing unmethylated CpG immunostimulatory sequences (ISS) as an adjuvant for an HIV-1 immunogen (inactivated, gp120-depleted HIV-1 virus particles). The addition of the ISS to HIV-1 in incomplete Freunds adjuvant (IFA) was the optimal combination for the production of HIV-1-specific immune responses as measured by IFN-gamma (p=0.002) and IgG antibody production. Furthermore, the group that was immunized with HIV/IFA/ISS, as well as the group that received HIV-1 in complete Freunds adjuvant (CFA), stimulated significantly more RANTES (a beta-chemokine) (p=0.002) production from lymph node cells. These results suggest that the addition of CpG immunostimulatory sequences to HIV antigens in IFA may optimize HIV-1-specific immune responses and provides further rationale for the testing of ISS in combination with gp120-depleted whole-killed HIV-1 in IFA as a potential preventive or therapeutic HIV-1 vaccine.

HIV-1-Specific CD4 helper function in persons with chronic HIV-1 infection on antiviral drug therapy as measured by ELISPOT after treatment with an inactivated, gp120-depleted HIV-1 in incomplete Freund's adjuvant.

Objective: We hypothesized that treatment of HIV‐1‐seropositive study subjects receiving potent antiviral therapy with an HIV‐specific immune‐based therapy would increase HIV‐1‐specific T‐helper immune function. Design: 10 HIV‐1‐seropositive study subjects receiving antiretroviral therapy were treated with an inactivated, gp120‐depleted immunogen in IFA (HIV‐1 immunogen, Remune) at baseline, week 12, and week 24. Methods: The frequency of HIV‐1 antigen‐stimulated interferon‐&ggr; (IFN‐&ggr;)producing cells was determined by the ELISPOT assay. Results: Study subjects significantly increased their frequency of HIV‐1‐stimulated (p < .001) or p24 antigen‐stimulated (p < .01) IFN‐&ggr;‐producing cells after one, two, and three treatments of HIV‐1 immunogen. Depletion of CD4 cells resulted in the strongest abrogation of the IFN‐&ggr; response. The frequency of HIV‐1 (r = 0.64; p = .0002) and p24 (r = 0.72; p < .001) antigen‐stimulated IFN‐&ggr;‐producing cells in the CD8‐depleted population before and after treatment was associated with the lymphocyte‐proliferative response. Conclusions: Treatment with HIV‐1 immunogen significantly enhanced the frequency of HIV‐1‐specific IFN‐&ggr;‐producing cells. Studies are ongoing to determine the relationship between this reversal of HIV‐specific anergy and virologic outcomes.

Phenotypic Analysis of Human Immunodeficiency Virus (HIV) Type 1 Cell-Mediated Immune Responses after Treatment with an HIV-1 Immunogen

It was hypothesized that immune recognition could be stimulated with combined immune-based and potent antiviral drug therapies. This study examined human immunodeficiency virus type 1 (HIV-1)-specific lymphocyte proliferation before and after treatment with an inactivated HIV-1 immunogen in 15 chronically infected HIV-1 seropositive subjects. Lymphocyte proliferation to the immunizing antigen (gp120-depleted HIV-1; P<.001), purified native p24 (P<.001), and recombinant p24 (P<.05) increased after treatment with the HIV-specific immune-based therapy. By HIV-1 antigen-specific flow cytometry, T helper CD4 lymphocytes, CD8 lymphocytes, and NK cells (all P<.001) were the predominant cell types proliferating in vitro after treatment. Additional phenotyping of proliferating cells revealed predominantly CD4 and CD8 memory (both P<.001) phenotypes. This study supports the concept that in vitro lymphocyte proliferation to HIV-1 antigens, augmented after treatment with an inactivated HIV-1 immunogen, involves primarily CD4 and CD8 cell memory immune responses.

HIV-Specific CD4+ and CD8+ Immune Responses Are Generated with a gp120-Depleted, Whole-Killed HIV-1 Immunogen with CpG Immunostimulatory Sequences of DNA

We examined the adjuvant effects of a synthetic CpG oligodeoxynucleotide immunostimulatory sequence (ISS) using a whole-killed, gp120-depleted HIV antigen (HIV-1 antigen) in a Lewis rat model. We hypothesized that HIV-1-specific CD4(+) T helper (Th) immune responses could be enhanced when an ISS was combined with an HIV-1 antigen in incomplete Freunds adjuvant (IFA). We also reasoned that if such Th responses were sufficient, such a combination might also induce HIV-specific CD8(+) T cell immune responses. Here we demonstrate that the HIV-1 antigen in IFA combined with ISS stimulates both CD4(+) and CD8(+) HIV-specific immune responses as measured by interferon-gamma (IFN-gamma) in the ELISPOT assay. A strong correlation between these CD4(+) and CD8(+) responses was demonstrated. Furthermore, we found that the HIV-1 antigen in IFA with ISS as an adjuvant stimulated strong antibody responses to core antigen (p24). These studies suggest that the combination of the whole-killed, gp120-depleted HIV-1 antigen in IFA with ISS may be an ideal candidate to test in nonhuman primates and in human studies as a preventive HIV-1 vaccine.

T-Helper-Cell Proliferative Responses to Whole-Killed Human Immunodeficiency Virus Type 1 (HIV-1) and p24 Antigens of Different Clades in HIV-1-Infected Subjects Vaccinated with HIV-1 Immunogen (Remune)

ABSTRACT The discovery of multiple subtypes of human immunodeficiency virus type 1 (HIV-1) worldwide has created new challenges for the development of both therapeutic and preventive AIDS vaccines. We examined T-helper proliferative responses to HIV-1 clade A, B, C, G, and E whole-killed virus and to HIV-1 clade G and B core (p24) antigens in HIV-1-infected subjects taking potent antiviral drugs who received HIV immunogen (Remune) therapeutic vaccination. Subjects who were immunized mounted strong proliferative responses to both whole virus and core antigens of the different clades. These results suggest that a whole-killed immunogen may have broad applications as a therapeutic as well as a preventive vaccine in the current multiclade HIV-1 pandemic.

Cell-mediated immune responses to autologous virus in HIV-1-seropositive individuals after treatment with an HIV-1 immunogen.

ObjectiveWe hypothesized that cell mediated immune responses to an HIV-1 immunogen (whole-killed, gp120-depleted HIV-1 in IFA, Remune) would include those to autologous virus. MethodsFive chronically HIV-1 infected individuals were examined for HIV-specific immune responses to their own virus (autologous viral antigen) after treatment with an HIV-1 immunogen. ResultsSubjects had low proliferative responses to HIV and p24 antigens prior to immunization and mounted strong lymphocyte proliferative responses to the immunizing HIV-1 virus, native p24, and autologous viral antigen post immunization. Similarly, subjects produced low amounts of interferon-γ in response to HIV and p24 antigens prior to immunization and increased their interferon-γ production in response to HIV-1, native p24, and to autologous antigen post-immunization. Furthermore, β-chemokine responses measured as migratory inhibitory protein-1β production were low at baseline in response to HIV-1 and native p24 antigens and were enhanced post immunization to HIV-1, native p24, and autologous antigen. ConclusionsIn this study HIV-specific immune responses to autologous virus were observed after treatment with an HIV-specific immunogen.

Maternal and newborn immunization with a human immunodeficiency virus-1 immunogen in a rodent model

We examined immunization with an inactivated, gp120‐depleted human immunodeficiency virus (HIV) antigen in incomplete Freunds adjuvant (IFA), also containing a sequence of immunostimulatory (ISS) DNA, during the last trimester of pregnancy and neonatally in a rat model. Pregnant rats were immunized in the third trimester and their litters were immunized during the newborn period. In addition, litters of rats from non‐immunized mothers were immunized during the neonatal period. As another control, pregnant rats were immunized and their litters analysed. Supernants from peripheral blood mononuclear cells (PBMCs) were assayed from newborns at 4 weeks of age for HIV‐specific interferon‐γ (IFN‐γ), HIV‐specific regulated on activation, normal, T‐cell expressed, and secreted (RANTES), and serum for p24 antigen‐specific immunoglobulin G (IgG) production. In the animals whose pregnant mothers were immunized and were also immunized during the neonatal period, we observed HIV‐specific IFN‐γ production and HIV‐specific RANTES production, but weak p24 IgG antibody production. Animals immunized only during the neonatal period developed the highest levels of HIV‐specific IFN‐γ production, but somewhat lower levels of HIV‐specific RANTES and p24 IgG antibody production. The group of animals whose mothers had received immunizations during the last trimester of pregnancy, but were not immunized during the neonatal period, developed the strongest p24 IgG antibody levels, but little or undetectable HIV‐specific IFN‐γ or RANTES production. Neonatal immunization resulted primarily in cell‐mediated immune responses, while animals born to mothers who were immunized during the last trimester had primarily an antibody‐mediated immune response. Immunization of pregnant animals followed by neonatal immunization resulted in a mixed cell‐mediated/antibody type profile in the neonatal animal. Future studies should provide insights into neonatal immunity and potential vaccine approaches to prevent neonatal infection and perinatal transmission.

The strategy of immunologic control of HIV-1 with structured treatment interruptions (STIs).

Structured treatment interruptions (STIs) have evolved as an experimental approach of interrupting highly active antiretroviral therapy (HAART) similar to cycles of cancer chemotherapy in order to develop immune control of HIV-1. There are multiple, ongoing clinical trials examining interruptions in antiviral therapy with and without immune-based therapies. If successful in developing immune control, STIs may be clinically useful but require large scale clinical trials to prove their utility and safety.