Harold Legg

Vaccination with HIV-1 gp120 DNA induces immune responses that are boosted by a recombinant gp120 protein subunit

Small animals were immunized with plasmid DNA encoding HIV-1 envelope gp120 either intramuscularly by needle injection (mice and guinea pigs) or epidermally with the Accell gene gun (guinea pits). Subsequently, the animals were boosted with a recombinant gp120 protein subunit vaccine in an oil-in-water based adjuvant, MF59. Antibodies and cytotoxic T-lymphocyte (CTL) immune responses to the HIV envelope glycoprotein were observed in animals immunized with gp120 DNA derived from the HIV-1SF2 laboratory strain or from HIV-1 field isolates. Titers of ELISA antibodies and serum neutralizing antibodies against the HIV-1SF2 laboratory isolate were substantially increased in DNA-immunized animals following a single boost with recombinant gp120 protein subunit. This DNA prime/protein subunit boost immunization approach may be important for vaccination against infectious agents such as HIV for which it is difficult to raise strong antiviral humoral responses with DNA vaccination alone.

An Alphavirus Replicon Particle Chimera Derived from Venezuelan Equine Encephalitis and Sindbis Viruses Is a Potent Gene-Based Vaccine Delivery Vector

ABSTRACT Alphavirus replicon particle-based vaccine vectors derived from Sindbis virus (SIN), Semliki Forest virus, and Venezuelan equine encephalitis virus (VEE) have been shown to induce robust antigen-specific cellular, humoral, and mucosal immune responses in many animal models of infectious disease and cancer. However, since little is known about the relative potencies among these different vectors, we compared the immunogenicity of replicon particle vectors derived from two very different parental alphaviruses, VEE and SIN, expressing a human immunodeficiency virus type 1 p55Gag antigen. Moreover, to explore the potential benefits of combining elements from different alphaviruses, we generated replicon particle chimeras of SIN and VEE. Two distinct strategies were used to produce particles with VEE-p55gag replicon RNA packaged within SIN envelope glycoproteins and SIN-p55gag replicon RNA within VEE envelope glycoproteins. Each replicon particle configuration induced Gag-specific CD8+ T-cell responses in murine models when administered alone or after priming with DNA. However, Gag-specific responses varied dramatically, with the strongest responses to this particular antigen correlating with the VEE replicon RNA, irrespective of the source of envelope glycoproteins. Comparing the replicons with respect to heterologous gene expression levels and sensitivity to alpha/beta interferon in cultured cells indicated that each might contribute to potency differences. This work shows that combining desirable elements from VEE and SIN into a replicon particle chimera may be a valuable approach toward the goal of developing vaccine vectors with optimal in vivo potency, ease of production, and safety.

Purification and Characterization of Oligomeric Envelope Glycoprotein from a Primary R5 Subtype B Human Immunodeficiency Virus

ABSTRACT Human immunodeficiency virus (HIV) continues to be a major public health problem throughout the world, with high levels of mortality and morbidity associated with AIDS. Considerable efforts to develop an effective vaccine for HIV have been directed towards the generation of cellular, humoral, and mucosal immune responses. A major emphasis of our work has been toward the evaluation of oligomeric (o-gp140) forms of the HIV type 1 (HIV-1) envelope protein for their ability to induce neutralizing antibody responses. We have derived stable CHO cell lines expressing o-gp140 envelope protein from the primary non-syncytium-inducing (R5) subtype B strain HIV-1US4. We have developed an efficient purification strategy to purify oligomers to near homogeneity. Using a combination of three detectors measuring intrinsic viscosity, light scattering, and refractive index, we calculated the molecular mass of the oligomer to be 474 kDa, consistent with either a trimer or a tetramer. The hydrodynamic radius (Rh ) of o-gp140 was determined to be 8.40 nm, compared with 5.07 nm for the monomer. The relatively smaller Rh of the oligomer suggests that there are indeed differences between the foldings of o-gp140 and gp120. To assess the structural integrity of the purified trimers, we performed a detailed characterization of the glycosylation profile of o-gp140, its ability to bind soluble CD4, and also its ability to bind to a panel of monoclonal antibodies with known epitope specificities for the CD4 binding site, the CD4 inducible site, the V3 loop, and gp41. Immunogenicity studies with rabbits indicated that the purified o-gp140 protein was highly immunogenic and induced high-titer, high-avidity antibodies directed predominantly against conformational epitopes. These observations confirm the structural integrity of purified o-gp140 and its potential as a vaccine antigen.

Antibody-Mediated Protection against Mucosal Simian-Human Immunodeficiency Virus Challenge of Macaques Immunized with Alphavirus Replicon Particles and Boosted with Trimeric Envelope Glycoprotein in MF59 Adjuvant

ABSTRACT We have previously shown that rhesus macaques were partially protected against high-dose intravenous challenge with simian-human immunodeficiency virus SHIVSF162P4 following sequential immunization with alphavirus replicon particles (VRP) of a chimeric recombinant VEE/SIN alphavirus (derived from Venezuelan equine encephalitis virus [VEE] and the Sindbis virus [SIN]) encoding human immunodeficiency virus type 1 HIV-1SF162 gp140ΔV2 envelope (Env) and trimeric Env protein in MF59 adjuvant (R. Xu, I. K. Srivastava, C. E. Greer, I. Zarkikh, Z. Kraft, L. Kuller, J. M. Polo, S. W. Barnett, and L. Stamatatos, AIDS Res. Hum. Retroviruses 22:1022-1030, 2006). The protection did not require T-cell immune responses directed toward simian immunodeficiency virus (SIV) Gag. We extend those findings here to demonstrate antibody-mediated protection against mucosal challenge in macaques using prime-boost regimens incorporating both intramuscular and mucosal routes of delivery. The macaques in the vaccination groups were primed with VRP and then boosted with Env protein in MF59 adjuvant, or they were given VRP intramuscular immunizations alone and then challenged with SHIVSF162P4 (intrarectal challenge). The results demonstrated that these vaccines were able to effectively protect the macaques to different degrees against subsequent mucosal SHIV challenge, but most noteworthy, all macaques that received the intramuscular VRP prime plus Env protein boost were completely protected. A statistically significant association was observed between the titer of virus neutralizing and binding antibodies as well as the avidity of anti-Env antibodies measured prechallenge and protection from infection. These results highlight the merit of the alphavirus replicon vector prime plus Env protein boost vaccine approach for the induction of protective antibody responses and are of particular relevance to advancing our understanding of the potential correlates of immune protection against HIV infection at a relevant mucosal portal of entry.

A Chimeric Alphavirus Replicon Particle Vaccine Expressing the Hemagglutinin and Fusion Proteins Protects Juvenile and Infant Rhesus Macaques from Measles

ABSTRACT Measles remains a major cause of child mortality, in part due to an inability to vaccinate young infants with the current live attenuated virus vaccine (LAV). To explore new approaches to infant vaccination, chimeric Venezuelan equine encephalitis/Sindbis virus (VEE/SIN) replicon particles were used to express the hemagglutinin (H) and fusion (F) proteins of measles virus (MV). Juvenile rhesus macaques vaccinated intradermally with a single dose of VEE/SIN expressing H or H and F proteins (VEE/SIN-H or VEE/SIN-H+F, respectively) developed high titers of MV-specific neutralizing antibody and gamma-interferon (IFN-γ)-producing T cells. Infant macaques vaccinated with two doses of VEE/SIN-H+F also developed neutralizing antibody and IFN-γ-producing T cells. Control animals were vaccinated with LAV or with a formalin-inactivated measles vaccine (FIMV). Neutralizing antibody remained above the protective level for more than 1 year after vaccination with VEE/SIN-H, VEE/SIN-H+F, or LAV. When challenged with wild-type MV 12 to 17 months after vaccination, all vaccinated juvenile and infant monkeys vaccinated with VEE/SIN-H, VEE/SIN-H+F, and LAV were protected from rash and viremia, while FIMV-vaccinated monkeys were not. Antibody was boosted by challenge in all groups. T-cell responses to challenge were biphasic, with peaks at 7 to 25 days and at 90 to 110 days in all groups, except for the LAV group. Recrudescent T-cell activity coincided with the presence of MV RNA in peripheral blood mononuclear cells. We conclude that VEE/SIN expressing H or H and F induces durable immune responses that protect from measles and offers a promising new approach for measles vaccination. The viral and immunological factors associated with long-term control of MV replication require further investigation.

Characterization of Human Immunodeficiency Virus Gag-Specific Gamma Interferon-Expressing Cells following Protective Mucosal Immunization with Alphavirus Replicon Particles

ABSTRACT A safe, replication-defective viral vector that can induce mucosal and systemic immune responses and confer protection against many infectious pathogens, such as human immunodeficiency virus type 1 (HIV-1), may be an ideal vaccine platform. Accordingly, we have generated and tested alphavirus replicon particles encoding HIV-1 Gag from Sindbis virus (SIN-Gag) and Venezuelan equine encephalitis virus (VEE-Gag), as well as chimeras between the two (VEE/SIN-Gag). Following intramuscular (i.m.), intranasal (i.n.), or intravaginal (IVAG) immunization with VEE/SIN-Gag and an IVAG challenge with vaccinia virus encoding HIV Gag (VV-Gag), a larger number of Gag-specific CD8+ intracellular gamma interferon-expressing cells (iIFNEC) were detected in iliac lymph nodes (ILN), which drain the vaginal/uterine mucosa (VUM), than were observed after immunizations with SIN-Gag. Moreover, a single i.n. or IVAG immunization with VEE/SIN-Gag induced a larger number of cells expressing HIV Gag in ILN, and immunizations with VEE/SIN-Gag through any route induced better protective responses than immunizations with SIN-Gag. In VUM, a larger percentage of iIFNEC expressed α4β7 or αEβ7 integrin than expressed CD62L integrin. However, in spleens (SP), a larger percentage of iIFNEC expressed α4β7 or CD62L than expressed αEβ7. Moreover, a larger percentage of iIFNEC expressed the chemokine receptor CCR5 in VUM and ILN than in SP. These results demonstrate a better induction of cellular and protective responses following immunizations with VEE/SIN-Gag than that following immunizations with SIN-Gag and also indicate a differential expression of homing and chemokine receptors on iIFNEC in mucosal effector and inductive sites versus systemic lymphoid tissues.

Immunogenicity of HIV-1 Env and Gag in baboons using a DNA prime/protein boost regimen.

Objectives: To evaluate the immunogenicity of sequence-modified HIV env and gag in baboons using DNA prime and protein boost strategy. Methods: Synthetic sequence-modified HIV gene cassettes were constructed that expressed three different forms of Env proteins, gp140, gp140mut and gp140TM, plus or minus a mutation in the protease-cleavage site. These plasmids were used to immunize baboons (Papio cynocephalus). A group of baboons was also immunized with both env and gag DNA followed by p55Gag virus-like particles (VLP) boost. Results: Modest antibody responses and low or no lymphoproliferative responses were observed following multiple DNA immunizations. In contrast, strong antibodies and substantial antigen-specific lymphoproliferative responses were seen following booster immunizations with oligomeric Env protein (o-gp140US4) in MF59. Neutralizing antibody responses were scored against T cell line adapted HIV-1 strains after the protein boosters, but neutralizing responses were low or absent against homologous and heterologous primary isolate strains. In the group receiving both gag and env vaccines, modest antigen-specific antibody and lymphoproliferative responses were scored after the DNA immunizations; these responses were enhanced several-fold upon boosting with the VLP preparations. The addition of Gag antigen did not interfere with Env-specific antibody responses, but there was a negative effect on the levels of Env-specific lymphoproliferation. Conclusions: These results highlight the importance of improving the potency of HIV DNA vaccines by enhanced DNA delivery and prime-boost vaccine technologies to generate more robust immune responses in larger animal models. In addition, care must be taken when immunizations with Env and Gag antigens are performed together.

Infection of baboons with a simian immunodeficiency virus/HIV-1 chimeric virus constructed with an HIV-1 Thai subtype E envelope

Objective:To construct an infectious chimeric simian immunodeficiency virus/HIV-1 (SHIV) with the envelope of a Thai subtype E HIV-1 strain for use in a non-human primate model. Methods:A novel SHIV genome was derived using the sequences of the ectodomain of the envelope gene from the Thai subtype E strain, HIV-19466. This SHIV (SHIV9466.33) was recovered by cocultivation from human peripheral blood mononuclear cells (PBMC) after transfection of human rhabdosarcoma cells. Rhesus macaque and baboon PBMC were screened in vitro for susceptibility to infection with SHIV9466.33. After successful infection of baboon PBMC, four animals were inoculated intravenously with SHIV9466.33 and monitored for plasma viral RNA, virus isolation from the PBMC, seroconversion, T-cell subsets, and signs of disease. Results:SHIV9466.33 was able to infect PBMC from 12 out of 14 baboons. All four of the baboons selected for in vivo inoculation became infected. Peak plasma viral RNA levels of 8000 to 700 000 RNA copies/ml were measured at 2 weeks post-inoculation. Virus was isolated from the PBMC of all four baboons during acute infection, and all seroconverted. Although transient declines in CD4+ T-cells were observed during early infection, CD4+ levels remained within normal ranges thereafter. In contrast, in vitro cultures of PBMC of four rhesus macaques were not susceptible to infection with SHIV9466.33. Conclusion:SHIV9466.33 containing an HIV-1 subtype E envelope displayed tropism for baboon PBMC but not for rhesus macaque PBMC. This chimeric virus established infection and induced antiviral antibodies in baboons inoculated by the intravenous route with cell-free virus. Thus, infection of baboons with SHIV9466.33 will serve as an important animal model for future studies of HIV-1 vaccine efficacy.

β7-integrin-independent enhancement of mucosal and systemic anti-HIV antibody responses following combined mucosal and systemic gene delivery

Vaccination strategies that can block or limit heterosexual human immunodeficiency virus (HIV) transmissions to local and systemic tissues are the goal of much research effort. Herein, in a mouse model, we aimed to determine whether the enhancement of antibody responses through mucosal and systemic immunizations, previously observed with protein‐based vaccines, applies to immunizations with DNA‐ or RNA‐based vectors. Intranasal (i.n.) followed by intramuscular (i.m.) immunizations (i.n./i.m.) with polylactide‐coglycolide (PLG)‐DNA microparticles encoding HIV‐gag (PLG‐DNA‐gag) significantly enhanced serum antibody responses, compared with i.m., i.n. or i.m. followed by i.n. (i.m./i.n.) immunizations. Moreover, while i.n./i.m., i.n. or i.m./i.n. immunizations with PLG‐DNA‐gag resulted in genital tract antibody responses, i.m. immunizations alone failed to do so. Importantly, β7‐deficient mice developed local and systemic antibody responses following i.n./i.m. immunization, or immunization via any other route, similar to those of wild‐type mice. To compare the DNA with an RNA delivery system, immunizations were performed with VEE/SIN‐gag replicon particles, composed of Venezuelan equine encephalitis virus (VEE) replicon RNA and Sindbis surface structure (SIN). i.n./i.m., compared with any other immunizations, i.n./i.m. immunization with VEE/SIN‐gag resulted in enhanced genital tract but not serum antibody responses. These data show for the first time that mucosal followed by systemic immunizations with gene delivery systems enhance B‐cell responses independent of the mucosal homing receptors α4β7 and αEβ7.

Lack of Interference with Immunogenicity of a Chimeric Alphavirus Replicon Particle-Based Influenza Vaccine by Preexisting Antivector Immunity

ABSTRACT Antivector immunity has been recognized as a potential caveat of using virus-based vaccines. In the present study, an alphavirus-based replicon particle vaccine platform, which has demonstrated robust immunogenicity in animal models, was tested for effects of antivector immunity on immunogenicity against hemagglutinin of influenza virus as a target antigen and efficacy for protection against lethal challenge with the virus. Chimeric alphavirus-based replicon particles, comprising Venezuelan equine encephalitis virus nonstructural and Sindbis virus structural components, induced efficient protective antibody responses, which were not adversely influenced after multiple immunizations with the same vector expressing various antigens.